Treatment cancers using a combination comprising btk inhibitors

ABSTRACT

Disclosed herein is a method for the prevention, delay of progression or treatment of cancer in a subject, comprising administering to the subject in need thereof a Btk inhibitor, particularly, (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetra-hydropyrazolo[1,5-a]pyrimidine-3-carboxamide or a pharmaceutically acceptable salt thereof, in combination with an immune checkpoint inhibitor or a targeted therapy agent. Also, disclosed a pharmaceutical combination comprising a Btk inhibitor, particularly, (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetra-hydropyrazolo[1,5-a]pyrimidine-3-carboxamide or a pharmaceutically acceptable salt thereof, in combination with an immune checkpoint inhibitor, or a targeted therapy agent and the use thereof.

This application claims the benefit of priority to InternationalApplication No. PCT/CN2016/096082 filed on Aug. 19, 2016, which ishereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

Disclosed herein is a method for the prevention, delay of progression ortreatment of cancer in a subject, comprising administering to thesubject in need thereof a Btk inhibitor (in particularly(S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo-[1,5-a]pyrimidine-3-carboxamideor a pharmaceutically acceptable salt thereof) in combination with animmune checkpoint inhibitor or a targeted therapy agent. Disclosedherein is also a pharmaceutical combination comprising a Btk inhibitor(in particularly(S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetra-hydropyrazolo[1,5-a]pyrimidine-3-carboxamideor a pharmaceutically acceptable salt thereof) in combination with animmune checkpoint inhibitor, or a targeted therapy agent and the usethereof.

BACKGROUND OF THE INVENTION

Bruton's tyrosine kinase (Btk) belongs to the Tec family of cytoplasmictyrosine kinases, which is the second largest family of non-receptorkinases in humans [Vetrie et al., Nature 361: 226-233, 1993; Bradshaw,Cell Signal. 22: 1175-84, 2010]. It is expressed in all cell lineages ofthe hematopoietic system, except for T cells and is localized in bonemarrow, spleen and lymph node tissue [Smith et al., J. Immunol. 152:557-565, 1994]. Inactivating mutations in the gene encoding Btk causeX-linked agammaglobulinemia (XLA) in humans and X-linkedimmunodeficiency (XID) in mice [Conley et al., Annu. Rev. Immunol. 27:199-227, 2009]. Both diseases are characterized by dramatic defects in Bcell development and function, suggesting the essential role of Btk forB cell development and function. In addition, constitutive activation ofBtk in B cells results in the accumulation of autoreactive plasma cells[Kersseboom et al., Eur J Immunol. 40:2643-2654, 2010]. Btk is activatedby upstream Src-family kinases in BCR signaling pahway. Once activated,Btk in turn phosphorylates phospholipase-Cy (PLCy), leading to Ca²⁺mobilization and activation of NF-κB and MAP kinase pathways. Theseproximal signaling events promote expression of genes involved inproliferation and survival [Humphries et al., J. Biol. Chem. 279: 37651,2004]. In addition to its essential regulatory role as downstream ofBCR, Btk activity also plays a critical role in FcR signaling. Signalingvia FcRγ associated receptors also promotes Btk-dependentproinflammatory cytokine production by cells such as macrophages [DiPaolo et al., Nat. Chem. Biol. 7: 41-50, 2011]. Btk has been animportant target due to its proximal location in the BCR and FcRsignaling pathways. Preclinical studies show that Btk deficient mice areresistant to developing collagen-induced arthritis. Moreover, clinicalstudies of Rittman, a CD20 antibody to deplete mature B-cells, revealthe key role of B-cells in a number of inflammatory diseases such asrheumatoid arthritis, systemic lupus erythematosus and multiplesclerosis [Gurcan et al., Int. Immunopharmacol. 9: 10-25, 2009]. Inaddition, aberrant activating of Btk plays important role inpathogenesis of B-cell lymphomas indicating that inhibition of Btk isuseful in the treatment of hematological malignancies [Davis et al.,Nature 463: 88-92, 2010].

In addition, aberrant activation of Btk plays an important role inpathogenesis of B-cell lymphomas indicating that inhibition of Btk isuseful in the treatment of hematological malignancies [Davis et al.,Nature 463: 88-92, 2010)]. The covalent BTK inhibitor ibrutinib(PCI-32765, Imbruvica®) was approved by the US Food and DrugAdministration for the treatment of chronic lymphocytic leukemia (CLL),mantle cell lymphoma (MCL) and Waldenstrom's macroglobulinemia (WM).

WO2014/173289A1 disclosed a series of fused heterocyclic compoundshaving the following general Formula (I) or a stereoisomer thereof, or apharmaceutically acceptable salt thereof as Btk inhibitors, which havedemonstrated potent inhibitory activity against Bruton's tyrosinekinase.

The unpublished PCT application PCT/CN2016/095510 disclosed acrystalline form of the Btk inhibitor in WO 2014/173289 A1,particularly,(S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetra-hydropyrazolo[1,5-a]pyrimidine-3-carboxamide(hereinafter Compound 1) for the treatment of cancers with aberrationsin the B-cell receptor (BCR) and FcR signaling pathway in which Btkplays important roles. Compound 1 has demonstrated to have potent andirreversible inhibitory activities against Btk.

The inventors of the present application have found that the combinationof a Btk inhibitor (in particular, the above-mentioned Compound 1) withan immunotherapy agent or a targeted therapy agent produces significantinhibition of tumor growth in cancers with aberrations in the B-cellreceptor as compared with the monotherapy of each of the above activepharmaceutical agent alone.

SUMMARY OF THE INVENTION

In a first aspect, disclosed herein is a method for the prevention,delay of progression or treatment of cancer in a subject, comprisingadministering to the subject in need thereof a therapeutically effectiveamount of a Btk inhibitor of Formula (I) or a stereoisomer thereof, or apharmaceutically acceptable salt thereof, in combination with atherapeutically effective amount of an immune checkpoint inhibitor or atargeted therapy agent.

In a second aspect, disclosed herein is a pharmaceutical combination foruse in the prevention, delay of progression or treatment of cancer,comprising a Btk inhibitor of Formula (I) or a stereoisomer thereof, ora pharmaceutically acceptable salt thereof, in combination with animmune checkpoint inhibitor or a targeted therapy agent.

In a third aspect, disclosed herein is a Btk inhibitor of Formula (I) ora stereoisomer thereof, or a pharmaceutically acceptable salt thereof,for use in the prevention, delay of progression or treatment of cancerin combination with an immune checkpoint inhibitor or a targeted therapyagent. In one embodiment of this aspect, disclosed herein is an immunecheckpoint inhibitor or a targeted therapy agent for use in theprevention, delay of progression or treatment of cancer in combinationwith a Btk inhibitor of Formula (I) or a stereoisomer thereof, or apharmaceutically acceptable salt thereof.

In a fourth aspect, disclosed herein is a use of a pharmaceuticalcombination in the manufacture of a medicament for use in theprevention, delay of progression or treatment of cancer, saidpharmaceutical combination comprising a Btk inhibitor of Formula (I) ora stereoisomer thereof, or a pharmaceutically acceptable salt thereof,and an immune checkpoint inhibitor or a targeted therapy agent.

In a fifth aspect, disclosed herein is an article of manufacture, or“kit” comprising a first container, a second container and a packageinsert, wherein the first container comprises at least one dose of amedicament comprising a Btk inhibitor of Formula (I) or a stereoisomerthereof, or a pharmaceutically acceptable salt thereof, the secondcontainer comprises at least one dose of a medicament comprising animmune checkpoint inhibitor or a targeted therapy agent, and the packageinsert comprises instructions for treating cancer a subject using themedicaments.

The method and pharmaceutical combination disclosed herein, as acombination therapy, produce significantly more efficacious than eithersingle agent.

In an embodiment of each of the above five aspects, the targeted therapyagent is an antibody. In an embodiment of each of the above fiveaspects, the targeted therapy agent is an anti-CD20 antibody. In someother embodiments of each of the above five aspects, the immunecheckpoint inhibitor is an antibody. In an embodiment of each of theabove five aspects, the immune check point inhibitor is an inhibitor ofPD-1, PD-L1, PD-L2, TIM-3, Gal-9, CTLA-4, CD80, CD86, A2AR, B7-H3,B7-H4, BTLA, HVEM, IDO1, IDO2, TDO, LAG3, VISTA, KIR, 2B4, CD2, CD27,CD28, CD30, CD40, CD90, CD137, CD226, DR³, GITR, ICOS, LAIR¹, LIGHT,MARCO, PS, OX-40, SLAM TIGHT, CTCNI, or any combinations thereof. In anembodiment of each of the above five aspects, the immune checkpointinhibitor is a monoclonal antibody. In an embodiments of each of theabove five aspects, the immune checkpoint inhibitor is an inhibitor ofPD-1.

In an embodiment of each of the above five aspects, the cancer is ahematologic cancer. In an embodiment of each of the above five aspects,the hematologic cancer is a leukemia, a lymphoma, a myeloma, anon-Hodgkin's lymphoma (NHL), a Hodgkin's lymphoma (HL), or a B-cellmalignancy. In an embodiments of each of the above five aspects, thehematologic cancer is a B-cell malignancy. In an embodiments of each ofthe above five aspects, the B-cell malignancy is chronic lymphocyticleukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma(FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL),Waldenstrom macroglobulinemia (WM), Hairy cell leukemia (HCL),Burkitt's-like leukemia (BL), B cell prolymphocytic leukemia (B-PLL),diffuse large B cell lymphoma (DLBCL), germinal center B-cell diffuselarge B-cell lymphoma (GCB-DLBCL), non-germinal center B-cell diffuselarge B-cell lymphoma (non-GCB DLBCL), DLBCL with undetermined subtype,primary central nervous system lymphoma (PCNSL), secondary centralnervous system lymphoma (SCNSL) of breast or testicular origin, multiplemyeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone Bcell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B celllymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblasticlarge cell lymphoma, precursor B-lymphoblastic lymphoma, B cellprolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginalzone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic)large B cell lymphoma, intravascular large B cell lymphoma, primaryeffusion lymphoma, lymphomatoid granulomatosis, or a combinationthereof. In an embodiment of each of the above five aspects, the B-cellmalignancy is diffuse large B-cell lymphoma (DLBCL). In an embodimentsof each of the above five aspects, DLBCL is activated B-cell diffuselarge B-cell lymphoma (ABC-DLBCL), GCB-DLBCL or Non-GCB DLBCL. In anembodiment of each of the above five aspects, the B-cell malignancy ischronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Bcell prolymphocytic leukemia (B-PLL), non-CLL/SLL lymphoma, follicularlymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL),Waldenstrom's macroglobulinemia (WM), multiple myeloma or a combinationthereof. In an embodiment of each of the above five aspects, the B-cellmalignancy is a relapsed or refractory (R/R) B-cell malignancy. In anembodiment of each of the above five aspects, the relapsed or refractoryB-cell malignancy is diffuse large B-cell lymphoma (DLBCL). In anembodiments of each of the above five aspects, the relapsed orrefractory DLBCL is activated B-cell diffuse large B-cell lymphoma(ABC-DLBCL), GCB-DLBCL or Non-GCB DLBCL. In an embodiment of each of theabove five aspects, the relapsed or refractory (R/R) B-cell malignancyis diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia(B-PLL), non-CLL/SLL lymphoma, follicular lymphoma (FL), mantle celllymphoma (MCL), marginal zone lymphoma (MZL), Waldenstrom'smacroglobulinemia (WM), multiple myeloma, or a combination thereof. Inan embodiment of each of the above five aspects, the B-cell malignancyis a metastasized B-cell malignancy. In an embodiment of each of theabove five aspects, the metastasized B-cell malignancy is diffuse largeB-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), smalllymphocytic lymphoma (SLL), B cell prolymphocytic leukemia (B-PLL),non-CLL/SLL lymphoma, follicular lymphoma (FL), mantle cell lymphoma(MCL), marginal zone lymphoma (MZL), Waldenstrom's macroglobulinemia(WM), multiple myeloma or a combination thereof.

In an embodiment of each of the above five aspects, the cancer is asarcoma, or carcinoma. In an embodiments of each of the above fiveaspects, the cancer is selected from anal cancer; appendix cancer; bileduct cancer (i.e., cholangiocarcinoma); bladder cancer; breast cancer;cervical cancer; colon cancer; cancer of Unknown Primary (CUP);esophageal cancer; eye cancer; fallopian tube cancer;gastroenterological cancer; kidney cancer; liver cancer; lung cancer;medulloblastoma; melanoma; oral cancer; ovarian cancer; pancreaticcancer; parathyroid disease; penile cancer; pituitary tumor; prostatecancer; rectal cancer; skin cancer; stomach cancer; testicular cancer;throat cancer; thyroid cancer; uterine cancer; vaginal cancer; vulvarcancer; or a combination thereof. In an embodiment of each of the abovefive aspects, the cancer is selected from bladder cancer, breast cancer,colon cancer, gastroenterological cancer, kidney cancer, lung cancer,ovarian cancer, pancreatic cancer, prostate cancer, proximal or distalbile duct cancer, melanoma, or a combination thereof. In an embodimentof each of the above five aspects, the colon cancer is adenocarcinoma,gastrointestinal carcinoid tumors, gastrointestinal stromal tumors,primary colorectal lymphoma, leiomyosarcoma, melanoma, squamouscell-carcinoma, mucinous adenocarcinoma, Signet ring celladenocarcinoma, or a combination thereof. In an embodiment of each ofthe above five aspects, the cancer is a relapsed or refractory cancer.In an embodiment of each of the above five aspects, the relapsed orrefractory cancer is selected from bladder cancer, breast cancer, coloncancer, gastroenterological cancer, kidney cancer, lung cancer, ovariancancer, pancreatic cancer, prostate cancer, proximal or distal bile ductcancer, melanoma, or a combination thereof. In an embodiment of each ofthe above five aspects, the cancer is a metastasized cancer. Inembodiments of each of the above five aspects, the metastasized canceris selected from bladder cancer, breast cancer, colon cancer,gastroenterological cancer, kidney cancer, lung cancer, ovarian cancer,pancreatic cancer, prostate cancer, proximal or distal bile duct cancer,and melanoma.

In an embodiment of each of the above five aspects, the BTK inhibitor is(S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetra-hydropyrazolo[1,5-a]pyrimidine-3-carboxamide(Compound 1), or a pharmaceutically acceptable salt thereof. In anembodiment of each of the above five aspects, the Btk inhibitor and theimmune checkpoint inhibitor, or a targeted therapy agent, areadministered simultaneously, sequentially or intermittently.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the combination effect of Compound 1 and anti-CD20 mAb(obinutuzumab) on tumor growth in REC-1/NK92MI MCL xenograft model.

FIG. 2 shows the combination effect of Compound 1 and anti-CD20 mAb(obinutuzumab) on tumor growth in human TMD-8 DLBCL xenograft model.

FIG. 3 shows the combination effects of anti-CD20 mAb (rituximab) andBTK inhibitor (including Compound 1 and ibrutinib) on tumor growth inhuman REC-1 MCL xenograft model.

FIG. 4 shows the combination effects of anti-CD20 mAb (rituximab) andBTK inhibitor (including Compound 1 and ibrutinib) on tumor weight inhuman REC-1 MCL xenograft model (on Day 14).

FIG. 5 shows the combination effects of anti-PD-1 mAb (Mab 1) andCompound 1 on tumor volume in human A431 epidermoid carcinoma allogeneicmodel.

FIG. 6 shows the combination effects of Compound 1 and anti-PD-1 mAb(pembrolizumab) on tumor volume in human A431 epidermoid carcinomaallogeneic model.

FIG. 7 shows the effect of BTK inhibitor (including Compound 1 andibrutinib) on anti-CD20 mAb (obinutuzumab) induced ADCC effect.

FIG. 8 shows an X-ray diffraction pattern of Compound 1 in a crystallineform.

FIG. 9 shows ¹H-NMR of the crystalline form of Compound 1.

FIG. 10 shows ¹³C-NMR of the crystalline form of Compound 1.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations (1):

ABC-DLBCL Activated B-cell diffuse large B-cell lymphoma A2AR AdenosineA2A receptor B-PLL B cell prolymphocytic leukemia Btk Bruton's TyrosineKinase BTLA B and T Lymphocyte Attenuator, CD272 CDR ComplementarityDetermining Region CLL chronic lymphocytic leukemia CTLA-4 CytotoxicT-Lymphocyte-Associated protein 4, CD152 DLBCL diffuse large B-celllymphoma DMEM Dulbecco minimum essential medium HVEM Herpesvirus EntryMediator non-CLL/SLL non-chronic lymphocytic leukemia/small lymphocyticlymphoma IDO Indoleamine 2,3-dioxygenase TDO Tryptophan 2,3-dioxygenaseIG immunoglobulin G i.p. Intraperitoneal or Intraperitoneally KIRKiller-cell Immunoglobulin-like Receptor LAG3 Lymphocyte ActivationGene-3 mAb Monoclonal antibodies NK Natural killer PBMC Peripheral bloodmononuclear cell PBS Phosphate Buffered Saline PD-1 Programmed Death 1protein, Pdcd-1, CD279 p.o. “by mouth” or “per os” QD Once daily Q4DOnce every four days QW Once weekly Q2W Once every two weeks Q3W Onceevery three weeks SLL small lymphocytic lymphoma TIM-3 T-cellImmunoglobulin domain and Mucin domain 3 Vh Heavy chain variable regionVl Light chain variable region VISTA V-domain Ig suppressor of T-cellactivation

Abbreviations (2):

ACN acetonitrile AcOH Acetic acid D-DBTA (2S, 3S)-Dibenzoyl tartaricacid DCM Dichloromethane DMF N,N-dimethylformamide DMF-DMAN,N-dimethylformamide dimethyl acetal EA Ethyl Acetate EDCI1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EtOAc ethyl acetate HOBtHydroxybenzotriazole HPLC High Performance Liquid Chromatography L-DBTA(2R, 3R)-Dibenzoyl tartaric acid MeCN Acetonitrile MeOH Methanol MeMgBrMethyl Magnesium Bromide MsOH Methanesulfonic Acid MTBE Methyl tertiarybutyl ether NLT not less than NMR Nuclear Magnetic Resonance NMT notmore than Pd Palladium pH Hydrogen ion concentration RT Room TemperatureTEA Triethylamine XRPD X-ray Powder Diffraction

Definitions

Unless specifically defined elsewhere in this document, all othertechnical and scientific terms used herein have the meaning commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs.

As used herein, including the appended claims, the singular forms ofwords such as “a”, “an”, and “the”, include their corresponding pluralreferences unless the context clearly indicates otherwise.

The term “or” is used to mean, and is used interchangeably with, theterm “and/or” unless the context clearly dictates otherwise.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof an active agent (e.g., a mAb or a Btk inhibitor) or a stated aminoacid sequence, but not the exclusion of any other active ingredient oramino acid sequence. When used herein the term “comprising” can beinterchangeable with the term “containing” or “including”.

The term “alkyl” refers to a hydrocarbon group selected from linear andbranched saturated hydrocarbon groups of 1-18, or 1-12, or 1-6 carbonatoms. Examples of the alkyl group include methyl, ethyl, 1-propyl orn-propyl (“n-Pr”), 2-propyl or isopropyl (“i-Pr”), 1-butyl or n-butyl(“n-Bu”), 2-methyl-1-propyl or isobutyl (“i-Bu”), 1-methylpropyl ors-butyl (“s-Bu”), and 1,1-dimethylethyl or t-butyl (“t-Bu”). Otherexamples of the alkyl group include 1-pentyl, 2-pentyl, 3-pentyl,2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl,1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl,2,3-dimethyl-2-butyl and 3,3-dimethyl-2-butyl groups. Lower alkyl means1-8, preferably 1-6, more preferably 1-4 carbon atoms; lower alkenyl oralkynyl means 2-8, 2-6 or 2-4 carbon atoms.

The term “alkenyl” refers to a hydrocarbon group selected from linearand branched hydrocarbon groups comprising at least one C═C double bondand of 2-18, or 2-12, or 2-6 carbon atoms. Examples of the alkenyl groupmay be selected from ethenyl or vinyl, prop-1-enyl, prop-2-enyl,2-methylprop-1-enyl, but-1-enyl, but-2-enyl, but-3-enyl,buta-1,3-dienyl, 2-methylbuta-1,3-diene, hex-1-enyl, hex-2-enyl,hex-3-enyl, hex-4-enyl, and hexa-1,3-dienyl groups.

The term “alkynyl” refers to a hydrocarbon group selected from linearand branched hydrocarbon group, comprising at least one CC triple bondand of 2-18, or 2-12, or 2-6 carbon atoms. Examples of the alkynyl groupinclude ethynyl, 1-propynyl, 2-propynyl (propargyl), 1-butynyl,2-butynyl, and 3-butynyl groups.

The term “cycloalkyl” refers to a hydrocarbon group selected fromsaturated and partially unsaturated cyclic hydrocarbon groups,comprising monocyclic and polycyclic (e.g., bicyclic and tricyclic)groups. For example, the cycloalkyl group may be of 3-12, or 3-8, or 3-6carbon atoms. Even further for example, the cycloalkyl group may be amonocyclic group of 3-12, or 3-8, or 3-6 carbon atoms. Examples of themonocyclic cycloalkyl group include cyclopropyl, cyclobutyl,cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl,cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cycloundecyl, and cyclododecyl groups. Examples of the bicycliccycloalkyl groups include those having 7-12 ring atoms arranged as abicycle ring selected from [4,4], [4,5], [5,5], [5,6] and [6,6] ringsystems, or as a bridged bicyclic ring selected frombicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, and bicyclo[3.2.2]nonane.The ring may be saturated or have at least one double bond (i.e.partially unsaturated), but is not fully conjugated, and is notaromatic, as aromatic is defined herein.

The term “Aryl” herein refers to a group selected from:5- and 6-memberedcarbocyclic aromatic rings, for example, phenyl; bicyclic ring systemssuch as 7-12 membered bicyclic ring systems wherein at least one ring iscarbocyclic and aromatic, selected, for example, from naphthalene, andindane; and tricyclic ring systems such as 10-15 membered tricyclic ringsystems wherein at least one ring is carbocyclic and aromatic, forexample, fluorene. For example, the aryl group is selected from 5- and6-membered carbocyclic aromatic rings fused to a 5- to 7-memberedcycloalkyl or heterocyclic ring optionally comprising at least oneheteroatom selected from N, O, and S, provided that the point ofattachment is at the carbocyclic aromatic ring when the carbocyclicaromatic ring is fused with a heterocyclic ring, and the point ofattachment can be at the carbocyclic aromatic ring or at the cycloalkylgroup when the carbocyclic aromatic ring is fused with a cycloalkylgroup. Bivalent radicals formed from substituted benzene derivatives andhaving the free valences at ring atoms are named as substitutedphenylene radicals. Bivalent radicals derived from univalent polycyclichydrocarbon radicals whose names end in “-yl” by removal of one hydrogenatom from the carbon atom with the free valence are named by adding“-idene” to the name of the corresponding univalent radical, e.g., anaphthyl group with two points of attachment is termed naphthylidene.Aryl, however, does not encompass or overlap with heteroaryl, separatelydefined below. Hence, if one or more carbocyclic aromatic rings arefused with a heterocyclic aromatic ring, the resulting ring system isheteroaryl, not aryl, as defined herein.

The term “halogen” or “halo” refers to F, Cl, Br or I.

The term “heteroalkyl” refers to alkyl comprising at least oneheteroatom.

The term “heteroaryl” refers to a group selected from: 5- to 7-memberedaromatic, monocyclic rings comprising 1, 2, 3 or 4 heteroatoms selectedfrom N, O, and S, with the remaining ring atoms being carbon; 8- to12-membered bicyclic rings comprising 1, 2, 3 or 4 heteroatoms, selectedfrom N, O, and S, with the remaining ring atoms being carbon and whereinat least one ring is aromatic and at least one heteroatom is present inthe aromatic ring; and 11- to 14-membered tricyclic rings comprising 1,2, 3 or 4 heteroatoms, selected from N, O, and S, with the remainingring atoms being carbon and wherein at least one ring is aromatic and atleast one heteroatom is present in an aromatic ring. For example, theheteroaryl group includes a 5- to 7-membered heterocyclic aromatic ringfused to a 5- to 7-membered cycloalkyl ring. For such fused, bicyclicheteroaryl ring systems wherein only one of the rings comprises at leastone heteroatom, the point of attachment may be at the heteroaromaticring or at the cycloalkyl ring. When the total number of S and O atomsin the heteroaryl group exceeds 1, those heteroatoms are not adjacent toone another. In some embodiments, the total number of S and O atoms inthe heteroaryl group is not more than 2. In some embodiments, the totalnumber of S and O atoms in the aromatic heterocycle is not more than 1.Examples of the heteroaryl group include, but are not limited to, (asnumbered from the linkage position assigned priority 1) pyridyl (such as2-pyridyl, 3-pyridyl, or 4-pyridyl), cinnolinyl, pyrazinyl,2,4-pyrimidinyl, 3,5-pyrimidinyl, 2,4-imidazolyl, imidazopyridinyl,isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, thiadiazolyl, tetrazolyl,thienyl, triazinyl, benzothienyl, furyl, benzofuryl, benzoimidazolyl,indolyl, isoindolyl, indolinyl, phthalazinyl, pyrazinyl, pyridazinyl,pyrrolyl, triazolyl, quinolinyl, isoquinolinyl, pyrazolyl,pyrrolopyridinyl (such as 1H-pyrrolo[2,3-b]pyridin-5-yl),pyrazolopyridinyl (such as 1H-pyrazolo[3,4-b]pyridin-5-yl), benzoxazolyl(such as benzo[d]oxazol-6-yl), pteridinyl, purinyl, 1-oxa-2,3-diazolyl,1-oxa-2,4-diazolyl, 1-oxa-2,5-diazolyl, 1-oxa-3,4-diazolyl,1-thia-2,3-diazolyl, 1-thia-2,4-diazolyl, 1-thia-2,5-diazolyl,1-thia-3,4-diazolyl, furazanyl, benzofurazanyl, benzothiophenyl,benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl,naphthyridinyl, furopyridinyl, benzothiazolyl (such asbenzo[d]thiazol-6-yl), indazolyl (such as 1H-indazol-5-yl) and5,6,7,8-tetrahydroisoquinoline.

The term “heterocyclic” or “heterocycle” or “heterocyclyl” refers to aring selected from 4- to 12-membered monocyclic, bicyclic and tricyclic,saturated and partially unsaturated rings comprising at least one carbonatoms in addition to 1, 2, 3 or 4 heteroatoms, selected from oxygen,sulfur, and nitrogen. “Heterocycle” also refers to a 5- to 7-memberedheterocyclic ring comprising at least one heteroatom selected from N, O,and S fused with 5-, 6-, and/or 7-membered cycloalkyl, carbocyclicaromatic or heteroaromatic ring, provided that the point of attachmentis at the heterocyclic ring when the heterocyclic ring is fused with acarbocyclic aromatic or a heteroaromatic ring, and that the point ofattachment can be at the cycloalkyl or heterocyclic ring when theheterocyclic ring is fused with cycloalkyl.

The “Heterocycle” also refers to an aliphatic spirocyclic ringcomprising at least one heteroatom selected from N, O, and S, providedthat the point of attachment is at the heterocyclic ring. The rings maybe saturated or have at least one double bond (i.e. partiallyunsaturated). The heterocycle may be substituted with oxo. The point ofthe attachment may be carbon or heteroatom in the heterocyclic ring. Aheterocyle is not a heteroaryl as defined herein. Examples of theheterocycle include, but not limited to, (as numbered from the linkageposition assigned priority 1) 1-pyrrolidinyl, 2-pyrrolidinyl,2,4-imidazolidinyl, 2,3-pyrazolidinyl, 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-piperidinyl, 2,5-piperazinyl, pyranyl, 2-morpholinyl,3-mo holinyl, oxiranyl, aziridinyl, thiiranyl, azetidinyl, oxetanyl,thietanyl, 1,2-dithietanyl, 1,3-difhietanyl, dihydropyridinyl,tetrahydropyridinyl, thiomorpholinyl, thioxanyl, piperazinyl,homopiperazinyl, homopiperidinyl, azepanyl, oxepanyl, thiepanyl,1,4-oxathianyl, 1,4-dioxepanyl, 1,4-oxafhiepanyl, 1,4-oxaazepanyl,1,4-dithiepanyl, 1,4-fhiazepanyl and 1,4-diazepane 1,4-dithianyl,1,4-azathianyl, oxazepinyl, diazepinyl, thiazepinyl, dihydrofhienyl,dihydropyranyl, dihydrofuranyl, tetrahydrofuranyl, tetrahydrothienyl,tetrahydropyranyl, tetrahydrothiopyranyl, 1-pyrrolinyl, 2-pyrrolinyl,3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, 1,4-dioxanyl,1,3-dioxolanyl, pyrazolinyl, pyrazolidinyl, dithianyl, difhiolanyl,pyrazolidinylimidazolinyl, pyrimidinonyl, 1,1-dioxo-thiomoholinyl,3-azabicyco[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl andazabicyclo[2.2.2]hexanyl. Substituted heterocycle also includes ringsystems substituted with one or more oxo moieties, such as piperidinylN-oxide, morpholinyl-N-oxide, 1-oxo-1-thiomorpholinyl and 1,1-dioxo-1-thiomorpholinyl.

Substituents are selected from: halogen, —R^(a), —OR^(a), ═O, ═NR^(a),═N—OR^(a), —NR^(a)R^(b), —SW, —SiR^(a)R^(a)R^(b), —OC(O)R^(a),—C(O)R^(a), —CO₂R^(a), —CONR^(a)R^(b), —OC(O)NR^(a)R^(b),—NR^(b)C(O)R^(a), —NR^(a)—C(O)NR^(b)R^(b), —NR^(a)—SO₂NR^(b),—NR^(b)CO₂R^(a), —NH—C(NH₂)═NH, —NR^(a)C(NH₂)═NH, —NH—C(NH₂)═NR^(a),—S(O)R^(a), —SO₂R^(a), —SO₂NR^(a)R^(b), —NR^(b)SO₂R, —CN and —NO₂, —N₃,—CH(Ph)₂, perfluoro(C₁-C₄)alkoxy and perfluoro(C₁-C₄)alkyl, in a numberranging from zero to three, with those groups having zero, one or twosubstituents being particularly preferred. R^(a), R^(b) and R^(c) eachindependently refer to hydrogen, unsubstituted (C₁-C₈)alkyl andheteroalkyl, unsubstituted aryl, aryl substituted with one to threehalogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, oraryl-(C₁-C₄)alkyl groups. When R^(a) and R^(b) are attached to the samenitrogen atom, they can be combined with the nitrogen atom to form a 5-,6- or 7-membered ring. Hence, —NR^(a)R^(b) includes 1-pyrrolidinyl and4-morpholinyl, “alkyl” includes groups such as trihaloalkyl (e.g., —CF₃and —CH₂CF₃), and when the aryl group is 1,2,3,4-tetrahydronaphthalene,it may be substituted with a substituted or unsubstituted(C₃-C₇)spirocycloalkyl group. The (C₃-C₇)spirocycloalkyl group may besubstituted in the same manner as defined herein for “cycloalkyl”.Preferred substituents are selected from: halogen, —R^(a), —OR^(a), ═O,—NR^(a)R^(b), —SR^(a), —SiR^(a)R^(a)R^(b), —OC(O)R^(a), —C(O)R^(a),—CO₂R^(a), —CONR^(a)R^(b), —OC(O)NR^(a)R^(b), —NR^(b)C(O)R^(a),—NR^(b)CO₂R^(a), —NR^(a)—SO₂NR^(b)R^(b), —S(O)R^(a), —SO₂R^(a),—SO₂NR^(a)R^(b), —NR^(b)SO₂R, —CN and —NO₂, perfluoro(C₁-C₄)alkoxy andperfluoro(C₁-C₄)alkyl, where R^(a) and R^(b) are as defined above.

The term “fused ring” herein refers to a polycyclic ring system, e.g., abicyclic or tricyclic ring system, in which two rings share only tworing atoms and one bond in common. Examples of fused rings may comprisea fused bicyclic cycloalkyl ring such as those having from 7 to 12 ringatoms arranged as a bicyclic ring selected from [4,4], [4,5], [5,5],[5,6] and [6,6] ring systems as mentioned above; a fused bicyclic arylring such as 7 to 12 membered bicyclic aryl ring systems as mentionedabove, a fused tricyclic aryl ring such as 10 to 15 membered tricyclicaryl ring systems mentioned above; a fused bicyclic heteroaryl ring suchas 8- to 12-membered bicyclic heteroaryl rings as mentioned above, afused tricyclic heteroaryl ring such as 11- to 14-membered tricyclicheteroaryl rings as mentioned above; and a fused bicyclic or tricyclicheterocyclyl ring as mentioned above.

When compounds contain olefin double bonds, unless specified otherwise,such double bonds are meant to include both E and Z geometric isomers.

Some of the compounds may exist with different points of attachment ofhydrogen, referred to as tautomers. For example, compounds includingcarbonyl —CH₂C(O)— groups (keto forms) may undergo tautomerism to formhydroxyl —CH═C(OH)— groups (enol forms). Both keto and enol forms,individually as well as mixtures thereof, are also intended to beincluded where applicable.

The term “Pharmaceutically acceptable salts” include, but are notlimited to salts with inorganic acids, selected, for example, fromhydrochlorates, phosphates, diphosphates, hydrobromates, sulfates,sulfinates, and nitrates; as well as salts with organic acids, selected,for example, from malates, maleates, fumarates, tartrates, succinates,citrates, lactates, methanesulfonates, p-toluenesulfonates,2-hydroxyethylsulfonates, benzoates, salicylates, stearates, alkanoatessuch as acetate, and salts with HOOC—(CH₂)_(n)—COOH, wherein n isselected from 0 to 4. Similarly, examples of pharmaceutically acceptablecations include, but are not limited to, sodium, potassium, calcium,aluminum, lithium, and ammonium.

In addition, if a compound is obtained as an acid addition salt, thefree base can be obtained by basifying a solution of the acid salt.Conversely, if the product is a free base, an addition salt, such as apharmaceutically acceptable addition salt, may be produced by dissolvingthe free base in a suitable organic solvent and treating the solutionwith an acid, in accordance with conventional procedures for preparingacid addition salts from base compounds. Those skilled in the art willrecognize various synthetic methodologies that may be used without undueexperimentation to prepare non-toxic pharmaceutically acceptableaddition salts.

The terms “administration”, “administering”, “treating” and “treatment”herein, when applied to an animal, human, experimental subject, cell,tissue, organ, or biological fluid, mean contact of an exogenouspharmaceutical, therapeutic, diagnostic agent, or composition to theanimal, human, subject, cell, tissue, organ, or biological fluid.Treatment of a cell encompasses contact of a reagent to the cell, aswell as contact of a reagent to a fluid, where the fluid is in contactwith the cell. The term “administration” and “treatment” also means invitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic,binding compound, or by another cell. The term “subject” herein includesany organism, preferably an animal, more preferably a mammal (e.g., rat,mouse, dog, cat, rabbit) and most preferably a human.

An “effective amount” refers to an amount of at least one compoundand/or at least one stereoisomer thereof, and/or at least onepharmaceutically acceptable salt thereof effective to “treat” a diseaseor disorder in a subject, and that will elicit, to some significantextent, the biological or medical response of a tissue, system, animalor human that is being sought, such as when administered, is sufficientto prevent development of, or alleviate to some extent, one or more ofthe symptoms of the condition or disorder being treated. Thetherapeutically effective amount will vary depending on the compound,the disease and its severity and the age, weight, etc., of the mammal tobe treated.

The term “at least one substituent” includes, for example, from 1 to 4,such as from 1 to 3, further as 1 or 2, substituents. For example, “atleast one substituent R¹⁶” herein includes from 1 to 4, such as from 1to 3, further as 1 or 2, substituents selected from the list of R<16> asdescribed herein.

The term “antibody” herein is used in the broadest sense andspecifically covers antibodies (including full length monoclonalantibodies) and antibody fragments so long as they recognize antigen,such as, a target antigen (e.g., CD20) or an immune checkpoint (e.g.,PD-1). An antibody molecule is usually monospecific, but may also bedescribed as idiospecific, heterospecific, or polyspecific. Antibodymolecules bind by means of specific binding sites to specific antigenicdeterminants or epitopes on antigens.

The term “monoclonal antibody” or “mAb” or “Mab” herein means apopulation of substantially homogeneous antibodies, i.e., the antibodymolecules comprised in the population are identical in amino acidsequence except for possible naturally occurring mutations that may bepresent in minor amounts. In contrast, conventional (polyclonal)antibody preparations typically include a multitude of differentantibodies having different amino acid sequences in their variabledomains, particularly their CDRs, which are often specific for differentepitopes. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. Monoclonal antibodies (mAbs) may beobtained by methods known to those skilled in the art. See, for example,U.S. Pat. No. 4,376,110. The mAbs disclosed herein may be of anyimmunoglobulin class including IgG, IgM, IgD, IgE, IgA, and any subclassthereof. A hybridoma producing a mAb may be cultivated in vitro or invivo. High titers of mAbs can be obtained in in vivo production wherecells from the individual hybridomas are injected intraperitoneally intomice, such as pristine-primed Balb/c mice to produce ascites fluidcontaining high concentrations of the desired mAbs. MAbs of isotype IgMor IgG may be purified from such ascites fluids, or from culturesupernatants, using column chromatography methods well known to those ofskill in the art.

In general, the basic antibody structural unit comprises a tetramer.Each tetramer includes two identical pairs of polypeptide chains, eachpair having one “light chain” (about 25 kDa) and one “heavy chain”(about 50-70 kDa). The amino-terminal portion of each chain includes avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The carboxy-terminal portion of theheavy chain may define a constant region primarily responsible foreffector function. Typically, human light chains are classified as kappaand lambda light chains. Furthermore, human heavy chains are typicallyclassified as α, δ, ε, γ, or μ and define the antibody's isotypes asIgA, IgD, IgE, IgG, and IgM, respectively. Within light and heavychains, the variable and constant regions are joined by a “J” region ofabout 12 or more amino acids, with the heavy chain also including a “D”region of about 10 more amino acids.

The variable regions of each light/heavy chain (V1/Vh) pair form theantibody binding site. Thus, in general, an intact antibody has twobinding sites. Except in bifunctional or bispecific antibodies, the twobinding sites are, in general, the same.

Typically, the variable domains of both the heavy and light chainscomprise three hypervariable regions, also called “complementaritydetermining regions (CDRs)”, which are located between relativelyconserved framework regions (FR). The CDRs are usually aligned by theframework regions, enabling binding to a specific epitope. In general,from N-terminal to C-terminal, both light and heavy chain variabledomains comprise FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2(CDR2), FR-3 (or FR3), CDR-3 (CDR3), and FR-4 (or FR4). The assignmentof amino acids to each domain is, generally, in accordance with thedefinitions of Sequences of Proteins of Immunological Interest, Kabat,et al., National Institutes of Health, Bethesda, Md.; 5<m> ed.; NIHPubl. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat,et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al, (1987) J.Mol. Biol. 196:901-917 or Chothia, et al, (1989) Nature 342:878-883.

The term “hypervariable region” means the amino acid residues of anantibody that are responsible for antigen-binding. The hypervariableregion comprises amino acid residues from a “complementarity determiningregion” or “CDR” (i.e., CDR-L1, CDR-L2 and CDR-L3 in the light chainvariable domain and CDR-H1, CDR-H2 and CDR-H3 in the heavy chainvariable domain). See, Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (defining the CDR regions of anantibody by sequence); see also Chothia and Lesk (1987) J. Mol. Biol.196: 901-917 (defining the CDR regions of an antibody by structure). Theterm “framework” or “FR” means those variable domain residues other thanthe hypervariable region residues defined herein as CDR residues.

Unless otherwise indicated, “antibody fragment” or “antigen-bindingfragment” means antigen binding fragments of antibodies, i.e. antibodyfragments that retain the ability to bind specifically to the antigenbound by the full-length antibody, e.g. fragments that retain one ormore CDR regions. Examples of antigen binding fragments include, but notlimited to, Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linearantibodies; single-chain antibody molecules, e.g., single chain Fv(ScFv); nanobodies and multispecific antibodies formed from antibodyfragments.

An antibody that “specifically binds to” a specified target protein isan antibody that exhibits preferential binding to that target ascompared to other proteins, but this specificity does not requireabsolute binding specificity. An antibody is considered “specific” forits intended target if its binding is determinative of the presence ofthe target protein in a sample, e.g. without producing undesired resultssuch as false positives. Antibodies or binding fragments thereof, usefulin the present invention will bind to the target protein with anaffinity that is at least two fold greater, preferably at least tentimes greater, more preferably at least 20-times greater, and mostpreferably at least 100-times greater than the affinity with non-targetproteins. An antibody herein is said to bind specifically to apolypeptide comprising a given amino acid sequence, e.g. the amino acidsequence of a mature human PD-1 molecule, if it binds to polypeptidescomprising that sequence but does not bind to proteins lacking thatsequence.

The term “human antibody” herein means an antibody that comprises humanimmunoglobulin protein sequences only. A human antibody may containmurine carbohydrate chains if produced in a mouse, in a mouse cell, orin a hybridoma derived from a mouse cell. Similarly, “mouse antibody” or“rat antibody” mean an antibody that comprises only mouse or ratimmunoglobulin protein sequences, respectively.

The term “humanized antibody” means forms of antibodies that containsequences from non-human (e.g., murine) antibodies as well as humanantibodies. Such antibodies contain minimal sequence derived fromnon-human immunoglobulin. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the hypervariable loopscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence. The humanized antibody optionally also will comprise at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. The prefix “hum”, “hu”, “Hu” or “h” is added toantibody clone designations when necessary to distinguish humanizedantibodies from parental rodent antibodies. The humanized forms ofrodent antibodies will generally comprise the same CDR sequences of theparental rodent antibodies, although certain amino acid substitutionsmay be included to increase affinity, increase stability of thehumanized antibody, or for other reasons.

The terms “cancer” or “tumor” herein mean or describe the physiologicalcondition involving abnormal cell growth with the potential to invade orspread to other parts of the body. The “disease” refers to any disease,discomfort, illness, symptoms or indications, and can be substitutedwith the term “disorder” or “condition”.

In some embodiments, the cancer is a hematologic cancer. In someembodiments, the hematologic cancer is a leukemia, a lymphoma, amyeloma, a non-Hodgkin's lymphoma (NHL), a Hodgkin's lymphoma (HL), or aB-cell malignancy. In some embodiments, the hematologic cancer is aB-cell malignancy. In some embodiments, the B-cell malignancy is chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicularlymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL),Waldenstrom macroglobulinemia (WM), Hairy cell leukemia (HCL),Burkitt's-like leukemia (BL), B cell prolymphocytic leukemia (B-PLL),diffuse large B cell lymphoma (DLBCL), germinal center B-cell diffuselarge B-cell lymphoma (GCB-DLBCL), non-germinal center B-cell diffuselarge B-cell lymphoma (non-GCB DLBCL), DLBCL with undetermined subtype,primary central nervous system lymphoma (PCNSL), secondary centralnervous system lymphoma (SCNSL) of breast or testicular origin, multiplemyeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone Bcell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B celllymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblasticlarge cell lymphoma, precursor B-lymphoblastic lymphoma, B cellprolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginalzone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic)large B cell lymphoma, intravascular large B cell lymphoma, primaryeffusion lymphoma, lymphomatoid granulomatosis, or a combinationthereof. In some embodiments, the B-cell malignancy is diffuse largeB-cell lymphoma (DLBCL). In some embodiments, DLBCL is activated B-celldiffuse large B-cell lymphoma (ABC-DLBCL), GCB-DLBCL or Non-GCB DLBCL.In some embodiments, the B-cell malignancy is chronic lymphocyticleukemia (CLL), small lymphocytic lymphoma (SLL), B cell prolymphocyticleukemia (B-PLL), non-CLL/SLL lymphoma, follicular lymphoma (FL), mantlecell lymphoma (MCL), marginal zone lymphoma (MZL), Waldenstrom'smacroglobulinemia (WM), multiple myeloma, or a combination thereof. Insome embodiments, the B-cell malignancy is a relapsed or refractory(R/R) B-cell malignancy. In some embodiments, the relapsed or refractory(R/R) B-cell malignancy is diffuse large B-cell lymphoma (DLBCL). Insome embodiments, the relapsed or refractory DLBCL is activated B-celldiffuse large B-cell lymphoma (ABC-DLBCL), GCB-DLBCL or Non-GCB DLBCL.In some embodiments, the relapsed or refractory (R/R) B-cell malignancyis chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL),B cell prolymphocytic leukemia (B-PLL), non-CLL/SLL lymphoma, follicularlymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL),Waldenstrom's macroglobulinemia (WM), multiple myeloma, or a combinationthereof. In some embodiments, the B-cell malignancy is a metastasizedB-cell malignancy. In some embodiments, the metastasized B-cellmalignancy is diffuse large B-cell lymphoma (DLBCL), chronic lymphocyticleukemia (CLL), small lymphocytic lymphoma (SLL), B cell prolymphocyticleukemia (B-PLL), non-CLL/SLL lymphoma, follicular lymphoma (FL), mantlecell lymphoma (MCL), marginal zone lymphoma (MZL), Waldenstrom'smacroglobulinemia (WM), multiple myeloma, or a combination thereof. Insome embodiments, the cancer is a sarcoma, or carcinoma. In someembodiments, the cancer is selected from anal cancer; appendix cancer;bile duct cancer (i.e., cholangiocarcinoma); bladder cancer; breastcancer; cervical cancer; colon cancer; cancer of Unknown Primary (CUP);esophageal cancer; eye cancer; fallopian tube cancer;gastroenterological cancer; kidney cancer; liver cancer; lung cancer;medulloblastoma; melanoma; oral cancer; ovarian cancer; pancreaticcancer; parathyroid disease; penile cancer; pituitary tumor; prostatecancer; rectal cancer; skin cancer; stomach cancer; testicular cancer;throat cancer; thyroid cancer; uterine cancer; vaginal cancer; or vulvarcancer; or a combination thereof. In some embodiments, the cancer isselected from bladder cancer, breast cancer, colon cancer,gastroenterological cancer, kidney cancer, lung cancer, ovarian cancer,pancreatic cancer, prostate cancer, proximal or distal bile duct cancer,melanoma, or a combination thereof. In some embodiments, the coloncancer is adenocarcinoma, gastrointestinal carcinoid tumors,gastrointestinal stromal tumors, primary colorectal lymphoma,leiomyosarcoma, melanoma, squamous cell-carcinoma, mucinousadenocarcinoma, Signet ring cell adenocarcinoma, or a combinationthereof. In some embodiments, the cancer is a relapsed or refractorycancer. In some embodiments, the relapsed or refractory cancer isselected from bladder cancer, breast cancer, colon cancer,gastroenterological cancer, kidney cancer, lung cancer, ovarian cancer,pancreatic cancer, prostate cancer, proximal or distal bile duct cancer,melanoma, or a combination thereof. In some embodiments, the cancer is ametastasized cancer. In embodiments, the metastasized cancer is selectedfrom bladder cancer, breast cancer, colon cancer, gastroenterologicalcancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer,prostate cancer, proximal or distal bile duct cancer, and melanoma.

The term “CDRs” means complementarity determining region(s) in animmunoglobulin variable region, defined using the Kabat numberingsystem, unless otherwise indicated.

Immune Checkpoint Inhibitors

In some embodiments, the Btk inhibitor is co-administered with an immunecheckpoint inhibitor, which is an inhibitor of PD-1, PD-L1, PD-L2,TIM-3, Gal-9, CTLA-4, CD80, CD86, A2AR, B7-H3, B7-H4, BTLA, BTLA, HVEM,IDO1, IDO2, TDO, LAG3, VISTA, KIR, 2B4, CD2, CD27, CD28, CD30, CD40,CD90, CD137, CD226, CD276, DR³, GITR, ICOS, LAIR¹, LIGHT, MARCO, PS,OX-40, SLAM TIGHT, CTCNI, or a combination thereof.

“Immune checkpoints (checkpoint proteins)” are molecules in the immunesystem that either turn up a signal (co-stimulatory molecules) or turndown a signal. And they also regulate T-cell activation or function.Many cancers protect themselves from the immune system by inhibiting theT cell signal. An “immune checkpoint inhibitor”, which totally orpartially reduces, inhibits, interferes with or modulates one or morecheckpoint proteins, has been increasing considered as targets forcancer immunotherapies. Numerous checkpoint proteins are known, such asPD-1 (Programmed Death 1, CD279) with its ligands PD-L1 (also namedCD274 or B7-H1) and PD-L2; TIM-3 (T-cell Immunoglobulin domain and Mucindomain 3, also known as HAVCR2) and its ligand Gal-9; CTLA-4 (CytotoxicT-Lymphocyte-Associated protein 4, CD152) and its ligands CD80 and CD86;and A2AR (Adenosine A2A receptor); B7-H3 (CD276); B7-H4 (VTCN1); BTLA (Band T Lymphocyte Attenuator, CD272) and its ligand HVEM (HerpesvirusEntry Mediator); IDO (Indoleamine 2,3-dioxygenase); LAG3 (LymphocyteActivation Gene-3); VISTA (V-domain Ig suppressor of T-cell activation);MR (Killer-cell Immunoglobulin-like Receptor). These proteins areresponsible for co-stimulatory or inhibitory interactions of T-cellresponses. Immune checkpoint proteins regulate and maintainself-tolerance and the duration and amplitude of physiological immuneresponses. Immune checkpoint inhibitors include antibodies or arederived from antibodies.

The immune system has multiple inhibitory pathways that are critical formaintaining self-tolerance and modulating immune responses. In T-cells,the amplitude and quality of response is initiated through antigenrecognition by the T-cell receptor and is regulated by immune checkpointproteins that balance co-stimulatory and inhibitory signals.

PD-1 is an immune checkpoint protein, that limits the activity of Tcells in peripheral tissues at the time of an inflammatory response toinfection and to limit autoimmunity PD-1 blockade in vitro enhancesT-cell proliferation and cytokine production in response to a challengeby specific antigen targets or by allogeneic cells in mixed lymphocytereactions. A strong correlation between PD-1 expression and response wasshown with blockade of PD-1 (Pardon, Nature Reviews Cancer, 12: 252-264,2012). PD-1 blockade can be accomplished by a variety of mechanismsincluding antibodies that bind PD-1 or its ligands. Examples of PD-1 andPD-L1 blockers, also named PD-1 and PD-L1 inhibitors, are described inU.S. Pat. Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149,and WO03042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342,WO2011159877, WO2011082400, WO2011161699, and WO2015035606. In someembodiments the PD-1 inhibitors include an antibody or a fragmentantigen binding thereof, which specifically binds to PD-1. In certainother embodiments the PD-1 blockers include anti-PD-1 antibodies andsimilar binding proteins such as nivolumab (MDX 1106, BMS 936558,ONO-4538, Opdivo®) described in U.S. Pat. No. 8,008,449B2, a fully humanIgG4 antibody that binds to and blocks the activation of PD-1 by itsligands PD-L1 and PD-L2; pembrolizumab (lambrolizumab, MK-3475 or SCH900475, Keytruda®) disclosed in U.S. Pat. No. 8,168,757B2, a humanizedmonoclonal IgG4 antibody against PD-1; pidilizumab (CT-011), a humanizedantibody that binds PD-1; AMP-224, a fusion protein of B7-DC; anantibody Fc portion; BMS-936559 (MDX-1105-01) for PD-L1 (B7-H1) blockadefor PD-1 blockade.

Other immune checkpoint protein is CTLA-4, that down-regulates pathwaysof T-cell activation (Fong et al., Cancer Res. 69(2):609-615, 2009;Weber Cancer Immunol. Immunother, 58:823-830, 2009). Blockade of CTLA-4has been shown to augment T-cell activation and proliferation.Inhibitors of CTLA-4 include anti-CTLA-4 antibodies. Anti-CTLA-4antibodies bind to CTLA-4 and block the interaction of CTLA-4 with itsligands CD80/CD86 expressed on antigen presenting cells and therebyblocking the negative down regulation of the immune responses elicitedby the interaction of these molecules. Examples of anti-CTLA-4antibodies are described in U.S. Pat. Nos. 5,811,097; 5,811,097;5,855,887; 6,051,227; 6,207,157; 6,682,736; 6,984,720; and 7,605,238.One anti-CDLA-4 antibody is tremelimumab (ticilimumab, CP-675,206). Inone embodiment, the anti-CTLA-4 antibody is ipilimumab (MDX-010,MDX-101, Yervoy®);) a fully human monoclonal IgG antibody that binds toCTLA-4. Ipilimumab is marketed under the name Yervoy™ and has beenapproved for the treatment of unresectable or metastatic melanoma. Otherimmune-checkpoint inhibitors include: LAG-3 inhibitors, such as IMP321,a soluble Ig fusion protein (Brignone et al., 2007, J. Immunol. 179:4202-4211); B7 inhibitors, such as B7-H3 and B7-H4 inhibitors, eg.,anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15(18) 3834); TIM3 inhibitors; A2AR inhibitors; BTLA inhibitors; IDOinhibitors, eg., INCB024360, an IDO1 inhibitor; VISTA inhibitors; or MRinhibitors, such as lirilumab (INN), an antibody binding to KIR²DL1/2L3.

In some embodiments, the immune checkpoint inhibitor is an antibody or afragment antigen binding thereof, or a chemical molecule drug. In someembodiments, the immune checkpoint inhibitor is a chemical moleculedrug, which is an inhibitor of PD-1, PD-L1, PD-L2, TIM-3, Gal-9, CTLA-4,CD80, CD86, A2AR, B7-H3, B7-H4, BTLA, BTLA, HVEM, IDO1, IDO2, TDO, LAG3,VISTA, MR, 2B4, CD2, CD27, CD28, CD30, CD40, CD90, CD137, CD226, CD276,DR³, GITR, ICOS, LAIR¹, LIGHT, MARCO, PS, OX-40, SLAM TIGHT, CTCNI, or acombination thereof; or an antibody or a fragment antigen bindingthereof, which specifically binds to one or more checkpoint proteinsselected from PD-1, PD-L1, PD-L2, TIM-3, Gal-9, CTLA-4, CD80, CD86,A2AR, B7-H3, B7-H4, BTLA, BTLA, HVEM, IDO1, IDO2, TDO, LAG3, VISTA, MR,2B4, CD2, CD27, CD28, CD30, CD40, CD90, CD137, CD226, CD276, DR³, GITR,ICOS, LAIR¹, LIGHT, MARCO, PS, OX-40, SLAM TIGHT, or CTCNI. In someembodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody.In some embodiments, the immune checkpoint is a monoclonal antibody. Insome embodiments, the immune checkpoint inhibitor is nivolumab orpidilizumab.

In some embodiments, the immune checkpoint inhibitor is a monoclonalantibody or a fragment thereof, disclosed in WO 2015/035606 A1.

Preferably, the anti-PD-1 monoclonal antibody is an antibody whichcomprises a heavy chain variable region (Vh) and a light chain variableregion (V1) that contain complement determinant regions (CDRs) listed asfollows:

a) mu317 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 11, 12, 13,respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 14, 15, 16,respectively); b) mu326 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 17, 18,19, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 20, 21,22, respectively); c) 317-4B6 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 31,32, 33, respectively); and CDR-L1 , CDR-L2 and CDR-L3 (SEQ ID NOs: 34,35, 36, respectively); d) 326-4A3 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs:37, 38, 39, respectively); and CDR-L1, CDR--L2 and CDR-L3 (SEQ ID NOs:40, 41, 42, respectively); e) 317-1H CDR-H1, CDR-H2 and CDR-H3 (SEQ IDNOs: 11, 59, 13, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ IDNOs: 14, 15, 16, respectively); f) 317-4B2 CDR-HL CDR-H2 and CDR-H3 (SEQID NOs: 11, 60, 13, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ IDNOs: 61, 15, 16, respectively); g)317-4B5 CDR-Hl, CDR-H2 and CDR-H3 (SEQID NOs: 11, 60, 13, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ IDNOs: 61, 15, 16, respectively); h)317-4B6 CDR-H1, CDR-H2 and CDR-H3 (SEQID NOs: 11, 32, 13, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ IDNOs: 61, 15, 16, respectively); i) 326-1 CDR-H1, CDR-H2 and CDR-H3 (SEQID NOs: 17, 62, 19, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ IDNOs: 20, 21, 22, respectively); j) 326-3B1 CDR-H1, CDR-H2 and CDR-H3(SEQ ID NOs: 17, 62, 19, respectively); and CDR-L1, CDR-L2 and CDR-L3(SEQ ID NOs: 20, 21, 22, respectively); or k) 326-3G1 CDR-H1, CDR-H2 andCDR-H3 (SEQ ID NOs: 17, 62, 19, respectively); and CDR-L1, CDR-I 2 andCDR-L3 (SEQ ID NOs: 20, 21, 22, respectively).

Preferably, the anti-PD-1 monoclonal antibody is an antibody whichcomprises a heavy chain variable region (Vh) and a light chain variableregion (V1) that contain any combinations of CDRs listed as follows:

(a) CDR-H1 (SEQ ID NO 31), CDR-H2 (SEQ ID NO 12, 32, 59 or 60) andCDR-H3 (SEQ ID NO 33), CDR-L1 (SEQ ID NO 14, 34 or 61), CDR-L2 (SEQ IDNO 35) and CDR-L3 (SEQ ID NO 36); or (b) CDR-H1 (SEQ ID NO 37), CDR-H2(SEQ ID NO 18, 38 or 62) and CDR-H3 (SEQ ID NO 39), CDR-L1 (SEQ ID NO40), CDR-L2 (SEQ ID NO 41) and CDR-L3 (SEQ ID NO 42).

Preferably, the anti-PD-1 monoclonal antibody is an antibody whichcomprises a heavy chain variable region (Vh) and a light chain variableregion (V1) comprising:

a) mu317 (SEQ ID NOs: 4 and 6, respectively); b) mu326 (SEQ ID NOs: 8and 10, respectively); c) 317-4B6 (SEQ ID NOs: 24 and 26, respectively);d) 326-4A3 (SEQ ID NOs: 28 and 30, respectively); e) 317-4B2 (SEQ IDNOs: 43 and 44, respectively); f) 317-4B5 (SEQ ID NOs: 45 and 46,respectively); g) 317-1 (SEQ ID NOs: 48 and 50, respectively); h)326-3B1 (SEQ ID NOs: 51 and 52, respectively); i) 326-3GI (SEQ ID NOs:53 and 54, respectively); j) 326-1 (SEQ ID NOs: 56 and 58,respectively); k) 317-3A1 (SEQ ID NOs: 64 and 26, respectively); l)317-3C1 (SEQ ID NOs: 65 and 26, respectively); m) 317-3E1 (SEQ ID NOs:66 and 26, respectively); n) 317-3F1 (SEQ ID NOs: 67 and 26,respectively); o) 317-3G1 (SEQ ID NOs: 68 and 26, respectively); p)317-3H1 (SEQ ID NOs: 69 and 26, respectively); q) 317-311 (SEQ ID NOs:70 and 26, respectively); r) 317-4B 1 (SEQ ID NOs: 71 and 26,respectively); s) 317-4B3 (SEQ ID NOs: 72 and 26, respectively); t)317-4B4 (SEQ ID NOs: 73 and 26, respectively); u) 317-4A2 (SEQ ID NOs:74 and 26, respectively); v) 326-3 A 1 (SEQ ID NOs: 75 and 30,respectively); w) 326-3C1 (SEQ ID NOs: 76 and 30, respectively); x)326-3D1 (SEQ ID NOs: 77 and 30, respectively); y) 326-3E1 (SEQ ID NOs:78 and 30, respectively); z) 326-3F1 (SEQ ID NOs: 79 and 30,respectively); aa) 326-3B N55D (SEQ ID NOs: 80 and 30, respectively);ab) 326-4A1 (SEQ ID NOs: 28 and 81, respectively); or ac) 326-4A2 (SEQID NOs: 28 and 82, respectively).

Preferably, the anti-PD-1 monoclonal antibody is an antibody whichcomprises a IgG4 heavy chain effector or constant domain comprising anyof SEQ ID NOs: 83-88.

Preferably, the anti-PD-1 monoclonal antibody is an antibody whichcontains a F(ab) or F(ab)2 comprising a domain said above, including aheavy chain variable region (Vh), a light chain variable region (V1) anda IgG4 heavy chain effector or constant domain.

Preferably, the anti-PD-1 monoclonal antibody is an antibody whichcomprise a heavy chain variable region (Vh) and a light chain variableregion (V1), and a IgG4 heavy chain effector or constant domaincomprising SEQ ID NOs: 87 or 88, wherein the heavy chain variable region(Vh) and the light chain variable region (V1) comprise:

a) mu317 (SEQ ID NOs: 4 and 6, respectively); b) mu326 (SEQ ID NOs: 8and 10, respectively); c) 317-4B6 (SEQ ID NOs: 24 and 26, respectively);d) 326-4A3 (SEQ ID NOs: 28 and 30, respectively); e) 317-4B2 (SEQ IDNOs: 43 and 44, respectively); f) 317-4B5 (SEQ ID NOs: 45 and 46,respectively); g) 317-1 (SEQ ID NOs: 48 and 50, respectively); h)326-3B1 (SEQ ID NOs: 51 and 52, respectively); i) 326-3GI (SEQ ID NOs:53 and 54, respectively); j) 326-1 (SEQ ID NOs: 56 and 58,respectively); k) 317-3A1 (SEQ ID NOs: 64 and 26, respectively); l)317-3C1 (SEQ ID NOs: 65 and 26, respectively); m) 317-3E1 (SEQ ID NOs:66 and 26, respectively); n)317-3F1 (SEQ ID NOs: 67 and 26,respectively); o) 317-3G1 (SEQ ID NOs: 68 and 26, respectively); p)317-3H1 (SEQ ID NOs: 69 and 26, respectively); q) 317-311 (SEQ ID NOs:70 and 26, respectively); r) 317-4B 1 (SEQ ID NOs: 71 and 26,respectively); s) 317-4B3 (SEQ ID NOs: 72 and 26, respectively); t)317-4B4 (SEQ ID NOs: 73 and 26, respectively); u) 317-4A2 (SEQ ID NOs:74 and 26, respectively); v) 326-3 A 1 (SEQ ID NOs: 75 and 30,respectively); w) 326-3C1 (SEQ ID NOs: 76 and 30, respectively); x)326-3D1 (SEQ ID NOs: 77 and 30, respectively); y) 326-3E1 (SEQ ID NOs:78 and 30, respectively); z) 326-3F1 (SEQ ID NOs: 79 and 30,respectively); aa) 326-3B N55D (SEQ ID NOs: 80 and 30, respectively);ab) 326-4A1 (SEQ ID NOs: 28 and 81, respectively); or ac) 326-4A2 (SEQID NOs: 28 and 82, respectively).

Preferably, the anti-PD-1 monoclonal antibody is an antibody whichcomprises a heavy chain variable region (Vh) and a light chain variableregion (V1) (comprising SEQ ID No 24 and SEQ ID No 26, respectively) anda IgG4 heavy chain effector or constant domain (comprising SEQ ID NO88), hereinafter Mab 1, which specifically binds to PD-1, especiallyPD-1 residues including K45 and 193; or, 193, L95 and P97, and inhibitsPD-1-medidated cellular signaling and activities in immune cells,antibodies binding to a set of amino acid residues required for itsligand binding.

The anti-PD1 monoclonal antibodies and antibody fragments thereof may beprepared in accordance with the disclosure of WO2015/035606 A1, theentire disclosure of which is expressly incorporated herein byreference. In a preferred embodiment, the anti-PD1 monoclonal antibodiesis Mab 1, which is administered at a dosage of about 2 mg/kg Q3W toabout 200 mg/kg Q3W.

Targeted Therapy Agent

In some embodiment, the Btk inhibitor is co-administered with antargeted therapy agent.

Targeted therapy is one of the major modalities of medical treatment forcancer, which blocks the growth of cancer cells by interfering withspecific targeted molecules needed for carcinogenesis and tumor growth,rather than by simply interfering with all rapidly dividing cells.B-lymphocyte antigen CD20 (CD20), which is an important target for thetreatment of B-cell malignancies, is an activated-glycosylatedphosphoprotein expressed on the surface of all B-cells beginning at thepro-B phase (CD45R+, CD117+) and progressively increasing inconcentration until maturity.

In some embodiments, the targeted therapy agent is an antibody. In someof the embodiments, the targeted therapy agent is an anti-CD20 antibody.In some of the embodiments, the targeted therapy agent is a monoclonalantibody. In some of the target therapy agent is selected fromrituximab, ibritumomab, tiuxetan, tositumonmab, ofatumumab orobinutuzumab.

Btk Inhibitors

“Btk inhibitor” means a compound of Formula (I), or a stereoisomerthereof, or a pharmaceutically acceptable salt thereof.

As disclosed in each of the above five aspects, the Btk inhibitor is acompound of Formula (I),

or a stereoisomer thereof, or a pharmaceutically acceptable saltthereof,wherein:

-   A is a 5- or 6-membered aromatic ring comprising 0-3 heteroatoms of    N, S or O;-   each W is independently —(CH₂)— or —C(O)—;-   L is a bond, CH₂, NR¹², O, or S;-   S/D is a single or double bond, and when a double bond, R⁵ and R⁷    are absent;-   m is 0, or an integer of 1-4;-   n is 0, or an integer of 1-4, wherein when n is more than 1, each R²    may be different;-   p is 0, or an integer of 1-2, wherein when p is 0, m is non-zero,    and when p is more than 1,-   each R⁶ and each R⁷ may be different;-   R¹, R⁴, R⁵, R⁶, and R⁷ are each independently H, halogen,    heteroalkyl, alkyl, alkenyl, cycloalkyl, aryl, saturated or    unsaturated heterocyclyl, heteroaryl, alkynyl, —CN, —NR¹³R¹⁴, —OR¹³,    —COR¹³, —CO₂R¹³, —CONR¹³R¹⁴, —C(═NR¹³)NR¹⁴R¹⁵, —NR¹³COR¹⁴,    —NR¹³CONR¹⁴R¹⁵, —NR¹³CO₂R¹⁴, —SO₂R¹³, —NR¹³SO₂NR¹⁴R¹⁵, or    —NR¹³SO₂R¹⁴, wherein the alkyl, alkenyl, alkynyl, cycloalkyl,    heteroaryl, aryl, and saturated or unsaturated heterocyclyl are    optionally substituted with at least one substituent R¹⁶, wherein    (R⁴ and R⁵), or (R⁴ and R⁶), or (R⁶ and R⁷), or (R⁶ and R⁶ when p is    2), together with the atoms to which they are attached, can form a    ring selected from cycloalkyl, saturated or unsaturated heterocycle,    aryl, and heteroaryl rings optionally substituted with at least one    substituent R¹⁶;-   R² is halogen, alkyl, —S-alkyl, —CN, —NR¹³R¹⁴, —OR¹³, —COR¹³,    —CO₂R¹³, —CONR¹³R¹⁴, —C(═NR¹³)NR¹⁴R¹⁵, —NR¹³COR¹⁴, —NR¹³CONR¹⁴R¹⁵,    —NR¹³CO₂R¹⁴, —SO₂R¹³, —NR¹³SO₂NR¹⁴R¹⁵, or —NR¹³SO₂R¹⁴;-   R¹² is H or lower alkyl;-   R¹³, R¹⁴ and R¹⁵ are each independently H, heteroalkyl, alkyl,    alkenyl, alkynyl, cycloalkyl, saturated or unsaturated heterocyclyl,    aryl, or heteroaryl; wherein (R¹³ and R¹⁴), and/or (R¹⁴ and R¹⁵)    together with the atom(s) to which they are attached, each can form    a ring selected from cycloalkyl, saturated or unsaturated    heterocycle, aryl, and heteroaryl rings optionally substituted with    at least one substituent R¹⁶; R¹⁶ is halogen, substituted or    unsubstituted alkyl, substituted or unsubstituted alkenyl,    substituted or unsubstituted alkynyl, substituted or unsubstituted    cycloalkyl, substituted or unsubstituted aryl, substituted or    unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl,    oxo, —CN, —OR^(a), —NR^(a)R^(b), —COR^(a), —CO₂R^(a),    —CONR^(a)R^(b), —C(═NR^(a))NR^(b)R^(c), —NR^(a)COR^(b),    —NR^(a)CONR^(a)R^(b), —NR^(a)CO₂R^(b), —SO₂R^(a), —SO₂aryl,    —NR^(a)SO₂NR^(b)R^(c), or —NR^(a)SO₂R^(b), wherein R^(a), R^(b), and    R^(c) are independently hydrogen, halogen, substituted or    unsubstituted alkyl, substituted or unsubstituted alkenyl,    substituted or unsubstituted alkynyl, substituted or unsubstituted    cycloalkyl, substituted or unsubstituted aryl, substituted or    unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl,    wherein (R^(a) and R^(b)), and/or (R^(b) and R^(c)) together with    the atoms to which they are attached, can form a ring selected from    cycloalkyl, saturated or unsaturated heterocycle, aryl, and    heteroaryl rings.

In some embodiments, the compound of Formula (I) is optically pure.

In some embodiments, S/D is a double bond and R⁵ and R⁷ are absent; p is1 and m is 0, 1 or 2; A is phenyl; and R⁴ and R⁶, together with theatoms to which they are attached, form a ring of formula

wherein Q is —CH₂—; J is —CH₂—; and d and b are each independently 0, oran integer of 1-4 S/D is a single bond; p is 1 and m is 0, 1 or 2; A isphenyl; or

-   S/D is a single bond; p is 0 and R⁶ and R⁷ are absent; A is phenyl.

In some embodiments, A is phenyl; W is —(CH₂)—; L is 0; S/D is a singlebond; m is 1; n is 0; p is 1; R¹ is phenyl; R² is absent; R⁵ is H; andR⁶ and R⁷ are H; yielding the combination structure:

In some embodiments, R⁴ is N-containing C₁-C₈ alkyl, N-containing C₃-C₈cycloalkyl and phenyl, each optionally substituted.

In some embodiments, R⁴ is methylamino, aniline group, azetidinyl,pyrrolidinyl, piperidinyl, azacycloheptenyl, each N-substituted with amoiety selected from benzyl, acyl, acryloyl, substituted-acryloyl,propiolyl, and substituted-propiolyl.

In some embodiments, R⁴ is selected from structures,

In some embodiments, R⁴ s l-acryloylpiperidin-4-yl (i.e., Compound 27 inWO 2014/173289 A1).

As disclosed in each of the above five aspects, the Btk inhibitor is acompound of Formula (II)—i.e., Compound 1,

or a pharmaceutically acceptable salt thereof.

The Btk inhibitor disclosed herein, such as the compound of Formula(II), may be synthesized by synthetic routes disclosed in WO 2014/173289A1 and unpublished PCT application PCT/CN2016/095510, the entiredisclosure of which is expressly incorporated herein by reference. TheBtk inhibitor, i.e., Compound 1, disclosed herein, may be prepared inaccordance with the procedures in PCT/CN2016/095510, the entiredisclosure of which is expressly incorporated herein by reference.

Combination Therapy

The combination therapy may be administered as a simultaneous, orseparate or sequential regimen. When administered sequentially, thecombination may be administered in two or more administrations. Thecombined administration includes co-administration, using separateformulation, and consecutive administration in either order, whereinpreferably there is a time period while both (or all) active agentssimultaneously exert their biological activities.

Suitable dosages for any of the above co-administered agents are thosepresently used and may be lowered due to the combined action (synergy)of the Btk inhibitor and the targeted therapy agent or the immunecheckpoint inhibitor, such as to increase the therapeutic index ormitigate toxicity or other side-effects or consequences.

In a particular embodiment of anti-cancer therapy, the Btk inhibitor andthe targeted therapy agent or the immune checkpoint inhibitor may befurther combined with surgical therapy and radiotherapy.

In an embodiment of each of the above five aspects, the amounts of theBtk inhibitor and the targeted therapy agent or the immune checkpointinhibitor disclosed herein and the relative timings of administration bedetermined by the individual needs of the patient to be treated,administration route, severity of disease or illness, dosing schedule,as well as evaluation and judgment of the designated doctor.

The Btk inhibitor and the targeted therapy agent or the immunecheckpoint inhibitor disclosed herein may be administered in variousknown manners, such as orally, topically, rectally, parenterally, byinhalation spray, or via an implanted reservoir, although the mostsuitable route in any given case will depend on the particular host, andnature and severity of the conditions for which the active ingredient isbeing administered. The term “parenteral” as used herein includessubcutaneous, intracutaneous, intravenous, intramuscular,intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,intralesional and intracranial injection or infusion techniques.

In one embodiment of each of the above five aspects, the Btk inhibitorand the targeted therapy agent or the immune checkpoint inhibitordisclosed herein may be administered in different route. In a preferredembodiment, the Btk inhibitor is administered orally, and the targetedtherapy agent or the immune checkpoint inhibitor is administeredparenterally such as subcutaneously, intracutaneously, intravenously orintraperitoneally. In a preferred embodiment, the BTK inhibitor isadministered once a day, two times per day, three times per day, fourtimes per day, or five times per day, and is administered at a dosage ofabout 80 mg/day to about 640 mg/day. In a preferred embodiment, the BTKinhibitor is administered at a dose of 320 mg QD or 160 mg BID.

EXAMPLE

The present invention is further exemplified, but not limited to, by thefollowing examples that illustrate the invention.

Example 1 Preparation of(S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide(Compound 1) and Crystalline Form A Thereof Step 1: Synthesis of BG-2

Under nitrogen atmosphere, to a solution of EA (5 v), HOBT (1.2 eq.),EDCI (1.2 eq.), 4-phenoxybenzoic acid (BG-1, 80 Kg, 1.0 eq.) andmalononitrile (1.2 eq.) was added TEA (2.4 eq.) at 10° C. The mixturewas then stirred at RT until the reaction was completed. The mixture wasthen centrifuged and the cake was washed with EA. The filtrate waswashed with aqueous NaHCO₃ twice and NH₄Cl. The organic phase was washedwith 1.5 N H₂SO₄ twice and stirred. Concentrated, precipitated frommethanol and purified water. The solid was collected by centrifugationand dried under vacuum. This gave 79.9 Kg of BG-2. ¹H NMR (DMSO-d₆) δ7.62 (d, J=8.6 Hz, 2H), 7.46-7.38 (m, 2H), 7.18 (t, J=7.4 Hz, 1H), 7.06(d, J=8.0 Hz, 2H), 6.94 (d, J=8.6 Hz, 2H).

Step 2: Synthesis of BG-3

Under nitrogen atmosphere, a solution of BG-2 (79.9 kg, 1.0 eq.) in MeCN(5.0 v) was added into trimethoxymethane (12.0 v) at 85° C. Theresultant mixture was stirred until the reaction was completed. Sampledfor HPLC analysis. Concentrated under vacuum. The residue wasprecipitated from i-PrOH and hexane. The mixture was centrifuged, andthe cake was washed with hexane and dried under vacuum. This gave 71.7Kg of product. ¹H NMR (400 MHz, DMSO-d₆) δ 7.70 (d, J=8.4 Hz, 2H),7.52-7.45 (m, 2H), 7.28 (t, J=7.6 Hz, 1H), 7.22-7.06 (m, 4H), 3.93 (s,3H).

Step 3: Synthesis of BG-4

Under nitrogen atmosphere, to a solution of BG-3 (71.6 kg, 1.0 eq.) inethanol (2.5 v) hydrazinium hydroxide (1.0 eq) in ethanol (0.6 v) wascharged dropwise to the reactor below 15° C. The solution was heated toRT and stirred until the reaction was completed. Water (4.0 v) was addedto the reactor. The solution was then cooled to 5° C., centrifuged andthe cake was washed with water (1.0 v). The cake was dried under vacuum.This gave 66.9 Kg of product. ¹H NMR (DMSO-d₆) δ 12.11 (br s, 1H), 7.80(d, J=8.8 Hz, 2H), 7.46-7.39 (m, 2H), 7.18 (t, J=7.6 Hz, 1H), 7.12-7.04(m, 4H), 6.43 (br s, 2H).

Steps 4 to 6: Synthesis of BG-8

To a mixture of DCM (8.0 v), BG-5 (80.0 Kg, 1.0 eq.),N,O-dimethylhydroxylamine hydrochloride (1.2 eq.), HOBt (1.2 eq.) andEDCI (1.2 eq.), TEA (2.6 eq.) was charged dropwise below 15° C. themixture was stirred at RT until the reaction was completed, centrifugedand the cake was washed with DCM (1.0 v) twice. The filtrate was washedwith 20% aqueous NH₄Cl (3×4.0 v). The filtrate was concentrated undervacuum to give the crude product BG-6, which was used in the next stepwithout further purification. The residue was dissolved in toluene (5.0v) and THF (1.0 v), cooled to 10° C., charged dropwise MeMgBr (1.4 eq.)at 10° C. and then stirred at RT until the reaction was completed. Thesolution was cooled below 10° C. Saturated aqueous NH₄Cl was chargeddropwise below 10° C. The mixture was centrifuged, separated, filtrated,and the organic phase was washed with aqueous NaCl twice. The organicphase was concentrated to give the crude product, which was used in thenext step without further purification. The residue in DMF (2.5 v) andDMF-DMA (2.5 v) was stirred at 110° C. until the reaction was completed.The reaction mixture was cooled, concentrated and then DCM was added.The final mixture was washed with saturated aqueous NH₄Cl. The organiclayer was concentrated and precipitated by charging hexane. The mixturewas centrifuged and the cake was collected. The cake was dried undervacuum. This gave 82.2 Kg of the desired product. ¹H NMR (DMSO-d₆) δ7.49 (d, J=12.6 Hz, 1H), 5.01 (d, J=12.6 Hz, 1H), 3.99-3.82 (m, 2H),3.14-2.94 (m, 2H), 2.89-2.61 (m, 6H), 2.49-2.37 (m, 1H), 1.66-1.56 (m,2H), 1.39 (s, 9H), 1.39-1.20 (m, 2H).

Step 7: Synthesis of BG-9

Under nitrogen atmosphere, a mixture of toluene (8.0 v), AcOH (0.5 v),BG-8 (1.2 eq.) and BG-4 (66.9 Kg 1.0 eq.) was heated to 95° C. andstirred until the reaction was completed. The mixture was cooled,concentrated and precipitated from methanol. The mixture was centrifugedand the cake was washed with methanol. The cake was dried under vacuum.This gave 107.8 Kg of product. ¹H NMR (DMSO-d₆) δ 8.78 (d, J=4.6 Hz,1H), 8.15-8.07 (m, 2H), 7.51-7.41 (m, 2H), 7.34 (d, J=4.6 Hz, 1H),7.27-7.19 (m, 3H), 7.17-7.10 (m, 2H), 4.24-4.02 (m, 2H), 3.81-3.69 (m,1H), 3.12-3.82 (m, 2H), 2.15-2.04 (m, 2H), 1.76-1.60 (m, 2H), 1.43 (s,9H).

Step 8: Synthesis of BG-10

To a mixture of THF (10.0 v), BG-9 (13.0 Kg, 1.0 eq.) and D-DBTA (1.0eq) under N₂ was charged Pd/C (10% w/w), hydrogen gas was introducedinto the reactor and the hydrogen pressure was maintained to 1.8 MPa.The reactor was heated to 40° C. slowly and stirred until the reactionwas completed. The mixture was then cooled, filtered, and the cake waswashed with THF. The filtrate was collected, and concentrated undervacuum. DCM was added. The residue was washed with aq. NaHCO₃,concentrated and precipitated from MTBE and hexane, then centrifuged.The cake was collected and dried under vacuum to give the desiredcompound (yield:94.8% and purity:98.5%). ¹H-NMR (DMSO-d₆) δ 7.82-7.76(m, 2H), 7.56-7.51 (m, 1H), 7.45-7.37 (m, 2H), 7.21-7.14 (m, 1H),7.12-7.03 (m, 4H), 4.09-3.91 (m, 3H), 3.30-3.22 (m, 2H), 2.82-2.55 (m,2H), 2.18-1.99 (m, 2H), 1.98-1.86 (m, 1H), 1.69-1.58 (m, 1H), 1.56-1.45(m, 1H), 1.38 (s, 9H), 1.32-1.13 (m, 2H).

Step 9: Synthesis of BG-11

To a solution of BG-10 (100.0 Kg 1.0 eq.) in DCM (6.0 v) was addeddropwise HCl in EtOH (20.9% w/w, 2.0 v) under nitrogen atmosphere. Themixture is stirred until the reaction was completed. MTBE (4.0 v) wasadded to the solution, cooled. The cakes was collected by centrifugationand washed with hexane (2.0 V), then the cake was slurried in hexane (5v), and centrifuged again. The cake was washed with hexane (2.0 V) anddried under vacuum. This gave 85.2 Kg product. ¹H-NMR (DMSO-d₆) δ9.25-8.85 (m, 2H), 7.84-7.70 (m, 2H), 7.47-7.37 (m, 2H), 7.18 (t, J=7.4Hz, 1H), 7.12-7.03 (m, 4H), 5.73 (br s, 2H), 4.12-4.03 (m, 1H),3.25-3.19 (m, 4H), 2.90-2.73 (m, 2H), 2.28-2.12 (m, 1H), 2.10-2.00 (m,1H), 1.99-1.86 (m, 1H), 1.84-1.52 (m, 4H).

Step 10: Synthesis of BG-11A

A mixture of BG-11 (85.0 Kg, 1.0 eq) in water (6.0 v) and NaOH (3.0 eq)was stirred until the reaction was completed at RT. The cake wascollected and slurried in MTBE (6.0 v). The mixture was then centrifugedto collect the cake. The cake was dried under vacuum. This gave 71.3 Kgproduct. ¹H-NMR (DMSO-d₆) δ 7.82-7.74 (m, 2H), 7.54-7.49 (m, 1H),7.45-7.38 (m, 2H), 7.21-7.14 (m, 1H), 7.12-7.04 (m, 4H), 4.03-3.95 (m,1H), 3.29-3.21 (m, 2H), 3.00-2.87 (m, 2H), 2.46-2.31 (m, 2H), 2.11-1.83(m, 3H), 1.58-1.12 (m, 4H).

Step 11: Synthesis of BG-11B

A mixture of enthanol/water/acetic acid (7:3:1, 46 v) and BG-11A (30 kg,1.0 eq.) in a reactor was heated to 70±5° C. under nitrogen atmosphere,then a solution of D-DBTA (1.20 eq.) in ethanol/water/acetic acid(7:3:1, 4 v) was added dropwise with the temperature not less than 65°C. The resulting solution was stirred for 16 hrs at 60-65° C., thencooled to RT. The solid was collected by centrifugation and washed withethanol (2.0 v). The cake was slurried in the mixed solvent ofethanol/water/AcOH (7:3:1, 20 v) for 16 hrs at 55° C. and cooled to RT.The solid was collected by centrifugation, washed with ethanol (2.0 v).The cake was dried under vacuum (Yield: 37.9%). ¹H-NMR (DMSO-d₆) δ 8.76(br s, 2H), 7.99-7.89 (m, 4H), 7.83-7.75 (m, 2H), 7.66-7.57 (m, 3H),7.52-7.45 (m, 4H), 7.45-7.39 (m, 2H), 7.21-7.14 (m, 1H), 7.13-7.03 (m,4H), 5.64 (s, 2H), 4.08-4.00 (m, 1H), 3.29-3.19 (m, 4H), 2.85-2.72 (m,2H), 2.21-1.40 (m, 7H).

Step 12: Synthesis of BG-11C

To a mixture of dichloromethane (15.0 v) and 20.0% aqueous KOH (3.0 v)was added bachwise BG-11B (48.0 kg, 1.0 eq.) under nitrogen atmosphereat RT. After the reaction was completed, the organic layer was collectedand the water layer was extracted with dichloromethane (5.0 v). Theorganic layers were combined. Con. HCl (0.36 v) was added to the aboveorganic layers at RT. The resulting mixture was stirred until thereaction was completed. The solid was collected by centrifugation andwashed with dichloromethane (1.0 v). The collected solid was slurriedwith MTBE (6.0 v). The solid was collected by centrifugation and washedwith MTBE (1.0 v), then was dried under vacuum. This gave 31.5 Kgproduct (Yield:100%).

Step 12: Synthesis of BG-11D (Alternative Intermediate)

ACN (5.0 v), soft water (10.0 v), KOH (5.0 eq) was charged to a reactorand stirred for at least 15 min. BG-11B (1.0 eq) was charge to thereactor in portion-wise. The mixture was stirred until the reaction wascompleted. The cake was collected by centrifugation, slurried in ACN(1.0 v) and soft water (5.0 v), and dried under vacuum to give theproduct.

Step 13: Synthesis of BG-12

A solution of BG-11C (15.0 Kg 1.0 eq.) in MsOH (2.5 v) was stirred at85° C. under nitrogen atmosphere until the reaction was completed. Aftercooling to 5° C. purified water (4.0 v) was added dropwise to the systemand kept the temperature not more than 35° C. (temperature increasedobviously). The resulting solution was stirred for 16 hrs at 30° C., andthen washed with DCM (2×3.0 v). The aqueous phase was collected. DCM(6.0 v) was added to the aqueous phase, the mixture was cooled to 5° C.The pH value was adjusted to 11˜12 with 20% aqueous NaOH (temperatureincreased obviously) with stirring with the temperature not more than30° C. The organic phase was separated and collected. The aqueous wasextracted with DCM (3.0 v). The organic layers were combined andconcentrated. MTBE (4.0 v) was added to the residue. The mixture wasthen concentrated and precipitated from n-heptane. The solid wascollected by centrifugation and dried in a vacuum oven. This gave 12.55Kg product (Yield: 94.9%). ¹H-NMR (DMSO-d₆) δ 7.52-7.46 (m, 2H),7.45-7.38 (m, 2H), 7.21-7.13 (m, 1H), 7.12-7.03 (m, 4H), 6.64 (s, 1H),3.99-3.90 (m, 1H), 3.29-3.22 (m, 2H), 3.03-2.90 (m, 2H), 2.48-2.36 (m,2H), 2.03 (dd, J=13.9, 5.6 Hz, 2H), 2.14-1.99 (m, 1H), 1.97-1.85 (m,1H), 1.65-1.15 (m, 3H).

Step 14: Synthesis of BG-13

A mixture of MeOH (13.5 v), purified water (4.5 v) and BG-12 (8.5 Kg,1.0 eq.) in a reactor was heated to 50° C. under N₂ atmosphere. To themixture was charged dropwise a solution of L-DBTA (0.7 eq) inMeOH/purified water (1.5 v/0.5 v) while keeping the temperature at 50°C. After addition, the mixture was stirred for at least 2 hrs at 50° C.,and then cooled to RT and stirred for at least 16 hrs at RT. The cakewas collected by Centrifugation and was washed with MeOH (2.0 v). Thecake was dried in a vacuum oven. This gave 9.08 Kg product (Yield:74.8%).

Step 15: Synthesis of(S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide(Compound 1)

Under N₂ atmosphere, ACN (12.0 v), water (12.5 v), BG-13 (8.0 Kg, 1.0eq), and NaHCO₃ (2.5 eq.) were added to a reactor. The mixture was thencooled to −5-0° C. To the mixture, the solution of acryloyl chloride(1.1 eq.) in MeCN (0.5 v) was added dropwise and stirred until thereaction was completed. EA (6.0 v) was then added to the reactor, andstirred. The organic phase was collected. The aqueous layer was furtherextracted with EA (3.0 v). The organic phases were combined and washedwith brine. The organic layer was collected and concentrated.

The residue was purified by silica gel (2 wt) column, eluted with 3% w/wmethanol in DCM (21.0 v). The Compound 1 solution was collected andconcentrated under vacuum. The residue was precipitated from EA/MTBE(2.0 v). The cake was collected by centrifugation as the product.

Step 15: Synthesis of(S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide(Compound 1, Alternative Method)

A mixture of CH₃CN (10.0 v), purified water (5.0 v), NaOH (1.5 eq.) andBG-13 (1.0 eq.) was stirred to get a clear solution. EtOAc (6.0 v) wasthen charged to the reaction and separated. The organic phase wascollected and washed with 15% brine (3.0 v) twice. The organic phaseprepared above was concentrated and the solvent was swapped to CH₃CN(residue volume: NMT 5.0 v). CH₃CN (7.5 v) and purified water (12.5 v)were charged and cooled to 15-20° C. L-(+)-tartaric acid (0.5 eq) andNaHCO₃ (2.5 eq.) were charged to the reaction mixture. A solution ofacryloyl chloride (1.1 eq.) in CH₃CN (0.5 v) was charged drop-wise tothe reaction mixture. After the reaction was completed, EtOAc (6.0 v)was charged to the reaction mixture and organic layer was collected.Aqueous phase was further extracted with EA (3.0 v). The organic layerswere combined, washed with 15% brine (5.0 v) and concentrated. Thesolvent was swapped to DCM (volume of residue: 1.5-2.0 v) and purifiedby silica gel column (silica gel: 100-200 mush, 2.0 w/w; eluent: 3% w/wMeOH in DCM (about 50 v). The collected solution was concentrated andswapped to EtOAc (4.0 v). MTBE (6.4 v) was charged drop-wise to residueat 50° C. The mixture was then cooled to 5° C. and the cake wascollected centrifugation.

Step 16: Preparation of Crystalline Form A of Compound 1

The above cake was dissolved in 7.0 volumes of DCM, and then swapped tosolvent EA. After recrystallization from EA/MTBE, the cakes wascollected by centrifugation, and was dried under vacuum. This gave 4.44Kg product (Yield: 70.2%).

The product was then characterized by X-ray powder diffraction (XRPD)pattern method, which was generated on a PANalytical Empyrean X-raypowder diffractometer with the XRPD parameters as follows: X-Raywavelength (Cu, kα, Kα1 (Å): 1.540598, Kα2 (Å): 1.544426; Kα2/Kα1intensity ratio: 0.50); X-Ray tube setting (45 Kv, 40 mA); divergenceslit (automatic); scan mode (Continuous); scan range (° 2TH) (3°-40);step size (° 2TH) (0.0131); scan speed (°/min) (about 10). The XRPDresult found the resultant product as a crystalline shown in FIG. 8.

The proton nuclear magnetic resonance (¹H-NMR) shown as in FIG. 9 wascollected on a Bruker 400M NMR Spectrometer in DMSO-d₆. ¹H-NMR (DMSO-d₆)δ 7.50 (d, J=8.6 Hz, 2H), 7.46-7.38 (m, 2H), 7.17 (t, J=7.6 Hz, 1H),7.08 (d, J=7.6 Hz, 2H), 7.05 (d, J=8.8 Hz, 2H), 6.85-6.72 (m, 1H), 6.67(s, 1H), 6.07 (dd, J=16.8, 2.2 Hz, 1H), 5.64 (dd, J=10.4 Hz, 2.2 Hz,1H), 4.55-4.38 (m, 1H), 4.17-3.94 (m, 2H), 3.33-3.22 (m, 2H), 3.08-2.88(m, 1H), 2.67-2.51 (m, 1H), 2.36-2.15 (m, 1H), 2.12-1.82 (m, 2H),1.79-1.65 (m, 1H), 1.63-1.49 (m, 1H), 1.38-1.08 (m, 2H).

The carbon nuclear magnetic resonance (¹³C-NMR) shown as in FIG. 10 wascollected on a Bruker 400M NMR Spectrometer in DMSO-d₆. ¹³C-NMR spectrafor Crystalline Form A of Compound 1.

Example 2 Effect of the Combination of Anti-CD20 mAb and BTK Inhibitorin Human REC-1/NK92MI Mantle Cell Lymphoma Xenograft Model

Method

REC-1 cells were cultured in RPMI1640 complete medium supplemented with10% (v/v) fetal bovine serum, and 100 μg/mL of penicillin andstreptomycin. NK92MI/CD16V cell line, overexpressing CD16 and FcRγchain, was established from parental cell line NK92MI. The cells weremaintained in MEM alpha supplemented with 0.2 mM inositol; 0.1 mM2-mercaptoethanol; 0.02 mM folic acid; 1×NEAA; adjust to a finalconcentration of 20% (v/v) fetal bovine serum and 100 μg/mL ofpenicillin and streptomycin. NOD/SCID mice were pre-treated withcyclophosphamide (prepared in saline, 150 mg/kg, i.p.) and disulfiram(prepared in 0.8% Tween-80 in saline, 125 mg/kg, p.o., one hour aftereach dose of cyclophosphamide) once daily for two days.

On the day of implantation, culture medium was replaced with freshmedium. Four hours later, media was removed and cells were collected asdescribed above. Cells were re-suspended in cold (4° C.) PBS and samevolume of matrigel was added to give a final concentration of 5×10⁷cells/mL for REC-1 and 1×10⁷ cells/mL for NK92MI/CD16V, respectively.Re-suspended cells were placed on ice prior to inoculation. The rightflank region of each mouse was cleaned with 75% ethanol prior to cellinoculation. Animals were then co-injected subcutaneously with 1×10⁷REC-1 cells and 2×10⁶NK92MI/CD16V cells in 200 μl of cell suspension inthe right front flank via a 26-gauge needle.

On day 3 after inoculation, animals were randomly divided into fourgroups with 12 mice per group. The groups consisted of a control group(no drug treatment), 2.5 mg/kg of Compound 1, 2 mg/kg of obinutuzumab(Gazyva®, obtained from Roch), and the combination of Compound 1 andobinutuzumab (2.5 mg/kg and 2 mg/kg, respectively). Treatments wereadministered in a volume of 10 mL/kg body weight, assessed immediatelybefore dosing and the volume dosed was adjusted accordingly. Compound 1was administered by oral gavage (p.o.) twice daily (BID) andobinutuzumab was administered by intraperitoneal (i.p.) injection onceweekly (QW). After implantation, primary tumor volume was measured intwo dimensions using a calliper.

Individual body weight was recorded twice weekly, with mice beingmonitored daily for clinical signs of toxicity for the duration of thestudy. Mice were euthanized using carbon dioxide when their tumor volumereached 2,500 mm³ after twice measurements, the tumor was ulcerated, orbody weight loss exceeded 20%.

Tumor volume was calculated using the formula: V=0.5×(a×b²) where a andb are the long and short diameters of the tumor, respectively. Tumorgrowth inhibition (TGI) was calculated using the following formula:

% TGI=100×[1−(treated_(t)/placebo_(t))]

-   -   treated_(t)=treated tumor volume at time t    -   placebo_(t)=placebo tumor volume at time t

Result

In vivo efficacy of Compound 1 and obinutuzumab was examined in humanREC-1/NK92MI MCL xenograft model. Compound 1, as a single agent, wasshown to be active in this model, with 72% TGI on day 23, whileobinutuzumab, as a single agent, was shown to have no anti-tumor effect.The combination of these two agents induced 98% TGI on day 23 which wassignificantly more efficacious than either single agent (FIG. 1).

Example 3 Effect of the Combination of Anti-CD20 mAb and BTK Inhibitorin Human TMD-8 DLBCL Xenograft Model Method

TMD-8 DLBCL cells were cultured in RPMI1640 complete medium supplementedwith 10% (v/v) fetal bovine serum, and 100 μg/mL of penicillin andstreptomycin. On the day of implantation, culture medium was replacedwith fresh medium. Four hours later, media was removed and cells werecollected as described above. Cells were re-suspended in cold (4° C.)PBS and same volume of matrigel was added to give a final concentrationof 5×10⁷ cells/mL for TMD-8 cells. Re-suspended cells were placed on iceprior to inoculation. The right flank region of each mouse was cleanedwith 75% ethanol prior to cell inoculation. Animals were then injectedsubcutaneously with 1×10⁷ TMD-8 cells in 200 μl of cell suspension inthe right front flank via a 26-gauge needle.

On day 7 after inoculation, animals were randomly divided into 4 groupswith 10 mice per group. The groups consisted of a control group (no drugtreatment), 7.5 mg/kg of Compound 1, 10 mg/kg of obinutuzumab (Gazyva®,obtained from Roch), and the combination of Compound 1 and obinutuzumab.Treatments were administered in a volume of 10 mL/kg body weight,assessed immediately before dosing and the volume dosed was adjustedaccordingly. Compound 1 was administered by oral gavage (p.o.) twicedaily (BID) and obinutuzumab was administered by intraperitoneal (i.p.)injection once every week (QW). After implantation, primary tumor volumewas measured in two dimensions using a calliper.

Mice were euthanized using carbon dioxide when their tumor volumereached 2,500 mm³ after twice measurements, the tumor was ulcerated, orbody weight loss exceeded 20%.

Tumor volume was calculated using the formula: V=0.5×(a×b²) where a andb are the long and short diameters of the tumor, respectively. Tumorgrowth inhibition (TGI) was calculated using the following formula:

% TGI=100×[1−(treated_(t)/placebo_(t))]

-   -   treated_(t)=treated tumor volume at time t    -   placebo_(t)=placebo tumor volume at time t

Result

On day 46, treatments of Compound 1 and obinutuzumab resulted in 81% and61% of tumor growth inhibition (TGI), respectively. Objective responseswere observed with Compound 1 at 7.5 mg/kg (1CR/4PR/5) and obinutuzumabat 10 mg/kg (OCR/2PR/2). The combination of Compound 1 and obinutuzumabwas more efficacious than each single agent treatment and resulted in111% TGI with 90% (1CR/8PR/9) overall response rate (FIG. 2).

Example 4 Effect of the Combination of Anti-CD20 mAb and BTK Inhibitorin Human REC-1 MCL Xenograft Model Method

REC-1 cells were cultured in RPMI1640 complete medium supplemented with10% (v/v) fetal bovine serum, and 100 μg/mL of penicillin andstreptomycin. On the day of implantation, culture medium was replacedwith fresh medium. Four hours later, media was removed and cells werecollected as described above. Cells were re-suspended in cold (4° C.)PBS and same volume of matrigel was added to give a final concentrationof 5×10⁷ cells/mL for REC-1. Re-suspended cells were placed on ice priorto inoculation. The right flank region of each mouse was cleaned with75% ethanol prior to cell inoculation. Animals were then injectedsubcutaneously with 1×10⁷ REC-1 cells in 200 μl of cell suspension inthe right front flank via a 26-gauge needle.

On day 9 after inoculation, animals were randomly divided into 6 groupswith 7 mice per group. The groups consisted of a control group (no drugtreatment), 25 mg/kg of Ibrutinib, 7.5 mg/kg of Compound 1, 200 μg/doseof rituximab, the combination of Compound 1 and rituximab (7.5 mg/kg and200 μg/dose, respectively), and the combination of Ibrutinib andrituximab (25 mg/kg and 200 μg/dose, respectively). Compound 1 andibrutinib were administered in a volume of 10 mL/kg body weight,assessed immediately before dosing and the volume dosed was adjustedaccordingly. Compound 1 and Ibrutinib were administered by oral gavage(p.o.) twice daily (BID) and rituximab was administered byintraperitoneal (i.p.) injection once every four days (Q4D). Afterimplantation, primary tumor volume was measured in two dimensions usinga calliper.

Individual body weight was recorded twice weekly, with mice beingmonitored daily for clinical signs of toxicity for the duration of thestudy. Mice were euthanized using carbon dioxide when their tumor volumereached 2,500 mm³ after twice measurements, the tumor was ulcerated, orbody weight loss exceeded 20%.

Tumor volume was calculated using the formula: V=0.5×(a×b²) where a andb are the long and short diameters of the tumor, respectively. Tumorgrowth inhibition (TGI) was calculated using the following formula:

% TGI=100×[1−(treated_(t)/placebo_(t))]

-   -   treated_(t)=treated tumor volume at time t    -   placebo_(t)=placebo tumor volume at time t

Result

As shown in FIG. 3, the tumor growth was retarded in rituximab, Compound1 and Ibrutinib treatment groups. Compound 1 achieved similar anti-tumoreffect as Ibrutinib, with 85% and 83% of TGI, respectively. Thecombination of Compound 1 and rituximab was more efficacious than eachsingle agent, whereas combination of Ibrutinib with rituximab showed noobvious combo effect. On day 14, Compound 1 in combination withrituximab caused significant lower tumor weight when compared withcombinational treatment of Ibrutinib and rituximab (FIG. 4).

Example 5 Effect of the Combination of anti-PD-1 mAb and Btk Inhibitorin Human A431 Epidermoid Carcinoma Allogeneic Model Method

On the day of implantation, human peripheral blood mononuclear cells(PBMCs) were isolated from 150 mL blood donated by a healthy volunteer.Briefly, peripheral blood was collected into vacuum blood collectiontubes containing sodium heparin. PBMCs were separated by densitygradient centrifugation using Histopaque-1077 and washed one time byDulbecco's Phosphate Buffered Saline (DPBS). The cell pellet wassuspended with DPBS at appropriate volume to give a final concentrationof 1×10⁸ cells/mL and placed on ice prior to inoculation.

A431 cells were cultured in DMEM complete medium supplemented with 10%(v/v) fetal bovine serum, and 100 μg/mL of penicillin and streptomycin.On the day of implantation, culture medium was replaced with freshmedium. Five hours later, media was removed and cells were collected asdescribed above, except that cells were re-suspended in cold (4° C.)DPBS to give a final concentration of 5×10⁷ cells/mL and placed on iceprior to inoculation. Mix the A431 cells, PBMCs and matrigel at theratio of 1:1:2. NOD/SCID mice were pre-treated with cyclophosphamide(prepared in saline, 150 mg/kg, i.p.) and disulfiram (prepared in 0.8%Tween-80 in saline, 125 mg/kg, p.o., one hour after each dose ofcyclophosphamide) once daily for one day. The right axilla region ofeach mouse was cleaned with 70% ethanol prior to cell inoculation. Eachanimal was injected subcutaneously with 2.5×10⁶ A431 cells and 5×10⁶PBMC (200 μl cell mixture in 50% matrigel) in the right front flank viaa 26-gauge needle 24 hours after the second dose of cyclophosphamide.

Starting from day 0 after cell inoculation, animals were randomlydivided into four groups with 10-11 mice per group. The groups consistedof a control group (no drug treatment), 15 mg/kg of Compound 1, 10 mg/kgof Mab 1, and the combination of Compound 1 and Mab 1 (15 mg/kg and 10mg/kg, respectively). Treatments were administered in a volume of 10mL/kg body weight, assessed immediately before dosing and the volumedosed was adjusted accordingly. Compound 1 was administered by oralgavage (p.o.) twice daily (BID) and Mab 1 was administered byintraperitoneal (i.p.) injection once weekly (QW). After implantation,primary tumor volume was measured in two dimensions using a calliper.

Individual body weight was recorded twice weekly, with mice beingmonitored daily for clinical signs of toxicity for the duration of thestudy. Mice were euthanized using carbon dioxide when their tumor volumereached 2,500 mm³, the tumor was ulcerated, or body weight loss exceeded20%.

Tumor volume was calculated using the formula: V=0.5×(a×b²) where a andb are the long and short diameters of the tumor, respectively.

Result

In vivo efficacy of Compound 1 and Mab 1 was examined in human A431epidermoid carcinoma allogeneic model. Mab 1, as a single agent, showedmarginal effect in this model while Compound 1 showed no anti-tumoreffect in this model. As shown in FIG. 5, the combination of these twoagents demonstrated better efficacy than either single agent.

Example 6 Effect of the Combination of Anti-PD-1 mAb and BTK Inhibitorin Human A431 Epidermoid Carcinoma Allogeneic Model Method

On the day of implantation, human peripheral blood mononuclear cells(PBMCs) were isolated from 150 mL blood donated by a healthy volunteer.Briefly, peripheral blood was collected into vacuum blood collectiontubes containing sodium heparin. PBMCs were separated by densitygradient centrifugation using Histopaque-1077 and washed one time byDulbecco's Phosphate Buffered Saline (DPBS). The cell pellet wassuspended with DPBS at appropriate volume to give a final concentrationof 5×10⁷ cells/mL and placed on ice prior to inoculation.

A431 cells were cultured in DMEM complete medium supplemented with 10%(v/v) fetal bovine serum, and 100 μg/mL of penicillin and streptomycin.On the day of implantation, culture medium was replaced with freshmedium. Five hours later, media was removed and cells were collected asdescribed above, except that cells were re-suspended in cold (4° C.)DPBS to give a final concentration of 5×10⁷ cells/mL and placed on iceprior to inoculation. Mix the A431 cells, PBMCs and matrigel at theratio of 1:1:2. NOD/SCID mice were pre-treated with cyclophosphamide(prepared in saline, 150 mg/kg, i.p.) and disulfiram (prepared in 0.8%Tween-80 in saline, 125 mg/kg, p.o., one hour after each dose ofcyclophosphamide) once daily for one day. The right axilla region ofeach mouse was cleaned with 70% ethanol prior to cell inoculation. Eachanimal was injected subcutaneously with 2.5×10⁶ A431 cells and 2.5×10⁶PBMC (200 μl cell mixture in 50% matrigel) in the right front flank viaa 26-gauge needle 24 hours after the second dose of cyclophosphamide.

Starting from day 0 after cell inoculation, animals were randomlydivided into four groups with 10-11 mice per group. The groups consistedof a control group (no drug treatment), 15 mg/kg of Compound 1, 10 mg/kgof pembrolizumab, and the combination of Compound 1 and pembrolizumab(15 mg/kg and 10 mg/kg, respectively). Treatments were administered in avolume of 10 mL/kg body weight, assessed immediately before dosing andthe volume dosed was adjusted accordingly. Compound 1 was administeredby oral gavage (p.o.) twice daily (BID) and pembrolizumab wasadministered by intraperitoneal (i.p.) injection once weekly (QW). Afterimplantation, primary tumor volume was measured in two dimensions usinga calliper.

Individual body weight was recorded twice weekly, with mice beingmonitored daily for clinical signs of toxicity for the duration of thestudy. Mice were euthanized using carbon dioxide when their tumor volumereached 2,500 mm³, the tumor was ulcerated, or body weight loss exceeded20%.

Tumor volume was calculated using the formula: V=0.5×(a×b²) where a andb are the long and short diameters of the tumor, respectively.

Result

In vivo efficacy of Compound 1 and pembrolizumab was examined in humanA431 epidermoid carcinoma allogeneic model. Neither Compound 1 norpembrolizumab showed anti-tumor effect in this model. As shown in FIG.6, the combination of these two agents showed no combo effect.

Example 7A Selectivity for BTK Against ITK of BTK Inhibitor Method:

Biochemical IC50 Determination of BTK and ITK

Compound 1 and Ibrutinib were tested for inhibition of BTK kinase inassays based on the time-resolved fluorescence-resonance energy transfer(TR-FRET) methodology. Briefly, the assays were carried out in 384-welllow volume black plates in a reaction mixture containing BTK kinase, 5μM ATP, 204 peptide substrate and 0-10 μM compound in buffer containing50 mM Tris pH7.4, 10 mM MgCl₂, 2 mM MnCl₂, 0.1 mM EDTA, 1 mM DTT, 0.005%Tween-20, 20 nM SEB and 0.01% BSA. The kinase was incubated withcompound for 60 minutes at room temperature and the reaction wasinitiated by the addition of ATP and peptide substrate. After reactionat room temperature for 60 minutes, an equal volume of stop/detectionsolution was added. Plates were sealed and incubated at room temperaturefor 1 hour, and the TR-FRET signals were recorded on a PHERAstar FSplate reader (BMG Labtech).

The protocol of ITK assay is similar to BTK assay except for thefollowing modification: 304 ATP and 204 TK substrate were used in thekinase reaction.

ITK p-PLCγ1 Cellular Assay

Once activated by T cell receptor (TCR) aggregation, ITK directlyphosphorylates PLCγ1 residue Tyr783 and subsequently actives NF-κBpathway, leading to increased production of Interleukin 2 (IL-2). Jurkatcells were treated with indicated concentration of ibrutinib or Compound1 for 2 hrs. After treatment, cells were exposed to 10 mM of hydrogenperoxide for 10 min. PLCγ1, p-PLCγ1 (Y783) were detected by western blotanalysis. IC50 is calculated using Quantity One and Prism 5 software.

ITK IL-2 Production Cellular Assay

HuT-78 cells and Hek293/OS8V cells were co-cultured in medium withindicated concentration of ibrutinib or Compound 1 for 20 hr. Potency ofibrutinib and Compound 1 were calculated basing on the medium IL-2level.

BTK Occupation Cellular Assay

Z-138 cells were treated with increasing concentration of Compound 1 for2 hours. The cell lysate were load to ELISA plate pre-immobilized withdetection probe. After overnight incubation, plate was washed with PBSTfor 3 times and probe conjugated BTK protein was detected by a BTKantibody. The potency of compounds was calculated basing on theinhibition of ratio between signal intensity at OD450 nm. IC50 valueswere calculated with GraphPad Prism software using the sigmoidaldose-response function.

Results:

In the biochemical assay, selectivity of Compound 1 for BTK was 187-foldagainst ITK, whereas selectivity of Ibrutinib for BTK was 17-foldagainst ITK, indicating that Compound 1 was more selective thanibrutinib for inhibition of BTK vs. ITK.

In the PLCγl phosphorylation assay (Jurkat cell) and IL-2 productionassay (HuT-78 cell), Compound 1 was shown to be 44-fold and 10-fold lesseffective than ibrutinib in inhibiting H₂O₂ induced PLCγ1phosphorylation and IL-2 production, suggesting that Compound 1 is muchweaker in ITK inhibition than ibrutinib.

TABLE 1 Compound 1 is highly selective for BTK against ITK IC₅₀ (nM)Targets Assays Ibrutinib Compound 1 BTK BTK cell based Occupation Assay2.3 2.2 BTK Biochemical Assay 0.18 0.3 ITK p-PLCγ1 Cellular Assay 773477 IL-2 production Cellular Assay 260 2536 ITK Biochemical Assay 3 56

Example 7B Effect of BTK Inhibitor on Anti-CD20 mAb Induced ADCC EffectMethod

Mino cells were cultured in RPMI1640 complete medium supplemented with10% (v/v) fetal bovine serum, and 100 μg/mL of penicillin andstreptomycin. NK92MI/CD16V cell line, overexpressing CD16 and FcRγchain, was established from parental cell line NK92MI. The cells weremaintained in MEM alpha supplemented with 0.2 mM inositol; 0.1 mM2-mercaptoethanol; 0.02 mM folic acid; 1×NEAA; adjust to a finalconcentration of 20% (v/v) fetal bovine serum and 100 μg/mL ofpenicillin and streptomycin.

Mino cells (2×10⁴ cells/well) as the target cells (T) were plated into96-well plate in triplicates. Cells were treated with vehicle or variousconcentrations of BTK inhibitors for one hour, followed by co-seedingwith NK92MI cells (4×10⁴ cells/well, as the effector cells (E)) andco-treatment with vehicle or obinutuzumab (2 ug/well) for additional 24hrs. After incubation, the cell-free supernatants of each well werecollected, and the levels of human IFN-γ were measured using Human IFN-γELISA Ready-SET-Go kit.

Result

Obinutuzumab-induced ADCC was assessed by IFN-γ secretion in co-cultureof NK cells and Mino cells (FIG. 7). Ibrutinib inhibited IFN-γ secretionin a dose-depended manner. At 1 μM, ibrutinib strongly inhibited IFN-γsecretion. In contrast, Compound 1 was much less effective in thisassay. At high dose tested (10 μM), it had similar inhibition effect onIFN-γ secretion as ibrutinib at 1 μM. The result suggest that Compound 1is at least 10-fold weaker than ibrutinib in inhibitingobinutuzumab-induced ADCC. This is consistent with Compound 1 being moreselective BTK inhibitor, with much weaker ITK inhibition activity thanibrutinib in both biochemical and cellular assays.

Example 8 Clinical Trial Phase Result on BTK Inhibitor Combined withAnti-CD20 Antibody

The multi-center, open-label Phase 1 trial of Compound 1 withobinutuzumab in patients with B-cell malignancies is being conducted inAustralia and the United States and consists of a dose-escalation phaseand a dose-expansion phase in disease-specific cohorts, which includetreatment naïve (TN) or relapsed/refractory (R/R) chronic lymphocytic(CLL)/small lymphocytic lymphoma (SLL) and R/R follicular lymphoma (FL).The dose-escalation component is testing Compound 1 at 320 mg once daily(QD) or 160 mg twice daily (BID) in 28-day cycles, in combination withobinutuzumab. And, obinutuzumab was administered in line with standardCLL dosing (three loading doses of 1000 mg weekly followed by 1000 mg onday one of cycles 2-6). The ongoing dose-expansion component is testingdoses of Compound 1 at 160 mg BID with the same obinutuzumab schedule.As of Mar. 31, 2017, 45 patients with CLL/SLL and 17 patients with FLwere enrolled in the trial.

43 patients with CLL/SLL (18 TN, 25R/R) and 15 patients with R/R FL hadgreater than 12 weeks of follow-up and were evaluable for efficacy. InTN CLL/SLL, after a median follow-up of 7.0 months (2.8-11.8 months),the overall response rate (ORR) was 89% with complete responses (CRs) in22% and partial responses (PRs) in 67% of patients. Stable disease (SD)was observed in 11% of patients. In R/R CLL/SLL, at a median follow-uptime of 8.0 months (3.8-14.0 months) the ORR was 92% with CRs in 16% andPRs in 76% of patients. SD was observed in 4% of patients. In R/R FL, ata median follow-up time of 6.2 months (1.2-10.7 months), the ORR was 73%with CRs in 33% and PRs in 40% of patients. Stable disease was observedin 13% of patients. One patient with R/R CLL/SLL had progressive disease(Richter's transformation), and two patients with R/R FL had progressivedisease.

The multi-center, open-label Phase 1 trial of Compound 1 withobinutuzumab in patients with B-cell malignancies is being conducted inAustralia and the United States and consists of a dose-escalation phaseand a dose-expansion phase in disease-specific cohorts, which includetreatment naïve (TN) or relapsed/refractory (R/R) chronic lymphocytic(CLL)/small lymphocytic lymphoma (SLL) and R/R follicular lymphoma (FL).The dose-escalation component is testing Compound 1 at 320 mg once daily(QD) or 160 mg twice daily (BID) in 28-day cycles, in combination withobinutuzumab. And, obinutuzumab was administered in line with standardCLL dosing (three loading doses of 1000 mg weekly followed by 1000 mg onday one of cycles 2-6). The ongoing dose-expansion component is testingdoses of Compound 1 at 160 mg BID with the same obinutuzumab schedule.As of Mar. 31, 2017, 45 patients with CLL/SLL and 17 patients with FLwere enrolled in the trial.

The foregoing examples and description of certain embodiments should betaken as illustrating, rather than as limiting the present invention asdefined by the claims. As will be readily appreciated, numerousvariations and combinations of the features set forth above can beutilized without departing from the present invention as set forth inthe claims. All such variations are intended to be included within thescope of the present invention. All references cited are incorporatedherein by reference in their entireties.

1. A method for the prevention, delay of progression or treatment ofcancer in a subject, comprising administering to the subject in needthereof a therapeutically effective amount of a Btk inhibitor, incombination with a therapeutically effective amount of an immunecheckpoint inhibitor, or a targeted therapy agent, wherein the Btkinhibitor is a compound of Formula (I),

or a stereoisomer thereof, or a pharmaceutically acceptable saltthereof, wherein: A is a 5- or 6-membered aromatic ring comprising 0-3heteroatoms of N, S or O; each W is independently —(CH₂)— or —C(O)—; Lis a bond, CH₂, NR¹², O, or S; S/D is a single or double bond, and whena double bond, R⁵ and R⁷ are absent; m is 0, or an integer of 1-4; n is0, or an integer of 1-4, wherein when n is more than 1, each R² may bedifferent; p is 0, or an integer of 1-2, wherein when p is 0, m isnon-zero, and when p is more than 1, each R⁶ and each R⁷ may bedifferent; R¹, R⁴, R⁵, R⁶, and R⁷ are each independently H, halogen,heteroalkyl, alkyl, alkenyl, cycloalkyl, aryl, saturated or unsaturatedheterocyclyl, heteroaryl, alkynyl, —CN, —NR₁₃R₁₄, —OR₁₃, —COR₁₃,—CO₂R₁₃, —CONR₁₃R₁₄, —C(═NR₁₃)NR₁₄R₁₅, —NR₁₃COR₁₄, —NR₁₃CONR₁₄R₁₅,—NR₁₃CO₂R₁₄, —SO₂R₁₃, —NR₁₃SO₂NR₁₄R₁₅, or —NR₁₃SO₂R₁₄, wherein thealkyl, alkenyl, alkynyl, cycloalkyl, heteroaryl, aryl, and saturated orunsaturated heterocyclyl are optionally substituted with at least onesubstituent R¹⁶, wherein (R⁴ and R⁵), or (R⁴ and R⁶), or (R⁶ and R⁷), or(R⁶ and R⁶ when p is 2), together with the atoms to which they areattached, can form a ring selected from cycloalkyl, saturated orunsaturated heterocycle, aryl, and heteroaryl rings optionallysubstituted with at least one substituent R¹⁶; R² is halogen, alkyl,—S-alkyl, —CN, —NR₁₃R₁₄, —OR¹³, —COR¹³, —CO₂R¹³, —CONR¹³R¹⁴,—C(═NR¹³)NR¹⁴R¹⁵, —NR¹³COR¹⁴, —NR¹³CONR¹⁴R¹⁵, —NR¹³CO₂R¹⁴, —SO₂R¹³,—NR¹³SO₂NR¹⁴R¹⁵, or —NR¹³SO₂R¹⁴; R¹² is H or lower alkyl; R¹³, R¹⁴ andR¹⁵ are each independently H, heteroalkyl, alkyl, alkenyl, alkynyl,cycloalkyl, saturated or unsaturated heterocyclyl, aryl, or heteroaryl;wherein (R¹³ and R¹⁴), and/or (R¹⁴ and R¹⁵) together with the atom(s) towhich they are attached, each can form a ring selected from cycloalkyl,saturated or unsaturated heterocycle, aryl, and heteroaryl ringsoptionally substituted with at least one substituent R¹⁶; R¹⁶ ishalogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheterocyclyl, oxo, —CN, —OR^(a), —NR^(a)R^(b), —COR^(a), —CO₂R^(a),—CONR^(a)R^(b), —C(═NR^(a))NR^(b)R^(c), —NR^(a)COR^(b),—NR^(a)CONR^(a)R^(b), —NR^(a)CO₂R^(b), —SO₂R^(a), —SO₂aryl,—NR^(a)SO₂NR^(b)R^(c), or —NR^(a)SO₂R^(b), wherein R^(a), R^(b), andR^(c) are independently hydrogen, halogen, substituted or unsubstitutedalkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted alkynyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocyclyl, wherein (R^(a)and R^(b)), and/or (R^(b) and R^(c)) together with the atoms to whichthey are attached, can form a ring selected from cycloalkyl, saturatedor unsaturated heterocycle, aryl, and heteroaryl rings.
 2. The method ofclaim 1, wherein the targeted therapy agent is an anti-CD20 antibody. 3.The method of claim 2, wherein the targeted therapy agent is anmonoclonal antibody.
 4. The method of claim 2, wherein the targetedtherapy agent is selected from rituximab, ibritumomab tiuxetan,tositumonmab, ofatumumab or obinutuzumab.
 5. The method of claim 1,wherein the immune checkpoint inhibitor is an inhibitor of PD-1,PD-PD-L1, PD-L2, TIM-3, Gal-9, CTLA-4, CD80, CD86, A2AR, B7-H3, B7-H4,BTLA, BTLA, HVEM, IDO1, IDO2, TDO, LAG3, VISTA, KIR, 2B4, CD2, CD27,CD28, CD30, CD40, CD90, CD137, CD226, CD276, DR³, GITR, ICOS, LAIR¹,LIGHT, MARCO, PS, OX-40, SLAM TIGHT, CTCNI, or a combination thereof. 6.The method of claim 5, wherein the immune checkpoint inhibitor is ananti-PD-1 antibody.
 7. The method of claim 6, wherein the immunecheckpoint inhibitor is a monoclonal antibody.
 8. The method of claim 6,wherein the immune checkpoint inhibitor is selected from nivolumab orpidilizumab.
 9. The method of claim 6, wherein the immune checkpointinhibitor is a monoclonal antibody or a fragment thereof, comprising aheavy chain variable region (Vh) amino acid sequence of SEQ ID No 24, alight chain variable region (V1) amino acid sequence of SEQ ID No 26,and a IgG4 constant domain amino acid sequence of SEQ ID NO
 88. 10. Themethod of claim 1, wherein the cancer is hematologic cancer.
 11. Themethod of claim 10, wherein the hematologic cancer is selected from aleukemia, a lymphoma, a myeloma, a non-Hodgkin's lymphoma (NHL), aHodgkin's lymphoma (HL), or a B-cell malignancy.
 12. The method of claim11, wherein the B-cell malignancy is chronic lymphocytic leukemia (CLL),small lymphocytic lymphoma (SLL), follicular lymphoma (FL), mantle celllymphoma (MCL), marginal zone lymphoma (MZL), Waldenstrommacroglobulinemia (WM), Hairy cell leukemia (HCL), Burkitt's-likeleukemia (BL), B cell prolymphocytic leukemia (B-PLL), diffuse large Bcell lymphoma (DLBCL), germinal center B-cell diffuse large B-celllymphoma (GCB-DLBCL), non-germinal center B-cell diffuse large B-celllymphoma (non-GCB DLBCL), DLBCL with undetermined subtype, primarycentral nervous system lymphoma (PCNSL), secondary central nervoussystem lymphoma (SCNSL) of breast or testicular origin, or a combinationof two or more thereof.
 13. The method of claim 12, wherein the diffuselarge B-cell lymphoma (DLBCL) is activated B-cell diffuse large B-celllymphoma (ABC-DLBCL), GCB-DLBCL or Non-GCB DLBCL.
 14. The method ofclaim 12, wherein the B-cell malignancy is selected form chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), B cellprolymphocytic leukemia (B-PLL), non-CLL/SLL lymphoma, follicularlymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL),Waldenstrom's macroglobulinemia (WM), multiple myeloma, or a combinationthereof.
 15. The method of any one of claims 12-14, wherein the B-cellmalignancy is a relapsed or refractory (R/R) B-cell malignancy.
 16. Themethod of claim 15, wherein the relapsed or refractory B-cell malignancyis diffuse large B-cell lymphoma (DLBCL).
 17. The method of claim 16,wherein the relapsed or refractory DLBCL is activated B-cell diffuselarge B-cell lymphoma (ABC-DLBCL), GCB-DLBCL or Non-GCB DLBCL.
 18. Themethod of claim 15, wherein the relapsed or refractory B-cell malignancyis selected form diffuse large B-cell lymphoma (DLBCL), chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), B cellprolymphocytic leukemia (B-PLL), non-CLL/SLL lymphoma, follicularlymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL),Waldenstrom's macroglobulinemia (WM), multiple myeloma, or a combinationthereof.
 19. The method of any one of the claims 12-14, wherein theB-cell malignancy is a metastasized B-cell malignancy.
 20. The method ofclaim 19, wherein the metastasized B-cell malignancy is selected formdiffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia(B-PLL), non-CLL/SLL lymphoma, follicular lymphoma (FL), mantle celllymphoma (MCL), marginal zone lymphoma (MZL), Waldenstrom'smacroglobulinemia (WM), multiple myeloma, or a combination thereof. 21.The method of claim 1, wherein the cancer is a sarcoma, or carcinoma.22. The method of claim 1 or 21, wherein the cancer is selected frombladder cancer, breast cancer, colon cancer, gastroenterological cancer,kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostatecancer, proximal or distal bile duct cancer, and melanoma.
 23. Themethod of claim 21, wherein the cancer is a relapsed or refractorycancer.
 24. The method of claim 23, wherein the relapsed or refractorycancer is selected from bladder cancer, breast cancer, colon cancer,gastroenterological cancer, kidney cancer, lung cancer, ovarian cancer,pancreatic cancer, prostate cancer, proximal or distal bile duct cancer,or melanoma.
 25. The method of any one of the claim 21, wherein thecancer is a metastasized cancer.
 26. The method of claim 25, wherein themetastasized cancer is selected from bladder cancer, breast cancer,colon cancer, gastroenterological cancer, kidney cancer, lung cancer,ovarian cancer, pancreatic cancer, prostate cancer, proximal or distalbile duct cancer, and melanoma.
 27. The method of claim 1, wherein theBTK inhibitor is(S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetra-hydropyrazolo[1,5-a]pyrimidine-3-carboxamide,or a pharmaceutically acceptable salt thereof.
 28. The method of claim1, wherein the BTK inhibitor administrated at a dose of 320 mg QD or 160mg BID.
 29. The method of claim 4, wherein the BTK inhibitor isadministrated at a dose of 320 mg QD or 160 mg BID in 28-day cycles, andobinutuzumab is administrated by three loading doses of 1000 mg weeklyfollowed by 1000 mg on day one of cycles 2-6.
 30. The method of claim 9,wherein the immune checkpoint inhibitor is administrated at a dose of 2mg/kg Q3W to 200 mg/kg Q3W.
 31. A pharmaceutical combination for use inthe prevention, delay of progression or treatment of cancer, comprisinga Btk inhibitor, and a targeted therapy agent, wherein the Btk inhibitoris a compound of Formula (I),

a stereoisomer thereof, or a pharmaceutically acceptable salt thereof,wherein: A is a 5- or 6-membered aromatic ring comprising 0-3heteroatoms of N, S or O; each W is independently —(CH₂)— or —C(O)—; Lis a bond, CH₂, NR¹², O, or S; S/D is a single or double bond, and whena double bond, R⁵ and R⁷ are absent; m is 0, or an integer of 1-4; n is0, or an integer of 1-4, wherein when n is more than 1, each R² may bedifferent; p is 0, or an integer of 1-2, wherein when p is 0, m isnon-zero, and when p is more than 1, each R⁶ and each R⁷ may bedifferent; R¹, R⁴, R⁵, R⁶, and R⁷ are each independently H, halogen,heteroalkyl, alkyl, alkenyl, cycloalkyl, aryl, saturated or unsaturatedheterocyclyl, heteroaryl, alkynyl, —CN, —NR¹³R¹⁴, —OR¹³, —COR¹³,—CO₂R¹³, —CONR¹³R¹⁴, —C(═NR¹³)NR¹⁴R¹⁵, —NR¹³COR¹⁴, —NR¹³CONR¹⁴R¹⁵,—NR¹³CO₂R¹⁴, —SO₂R¹³, —NR¹³SO₂NR¹⁴R¹⁵, or —NR¹³SO₂R¹⁴, wherein thealkyl, alkenyl, alkynyl, cycloalkyl, heteroaryl, aryl, and saturated orunsaturated heterocyclyl are optionally substituted with at least onesubstituent R¹⁶, wherein (R⁴ and R⁵), or (R⁴ and R⁶), or (R⁶ and R⁷), or(R⁶ and R⁶ when p is 2), together with the atoms to which they areattached, can form a ring selected from cycloalkyl, saturated orunsaturated heterocycle, aryl, and heteroaryl rings optionallysubstituted with at least one substituent R¹⁶; R² is halogen, alkyl,—S-alkyl, —CN, —NR¹³R¹⁴, —OR¹³, —COR¹³, —CO₂R¹³, —CONR¹³R¹⁴,—C(═NR¹³)NR¹⁴R¹⁵, —NR¹³COR¹⁴, —NR¹³CONR¹⁴R¹⁵, —NR¹³CO₂R¹⁴, —SO₂R¹³,—NR¹³SO₂NR¹⁴R¹⁵, or —NR¹³SO₂R¹⁴; R¹² is H or lower alkyl; R¹³, R¹⁴ andR¹⁵ are each independently H, heteroalkyl, alkyl, alkenyl, alkynyl,cycloalkyl, saturated or unsaturated heterocyclyl, aryl, or heteroaryl;wherein (R¹³ and R¹⁴), and/or (R¹⁴ and R¹⁵) together with the atom(s) towhich they are attached, each can form a ring selected from cycloalkyl,saturated or unsaturated heterocycle, aryl, and heteroaryl ringsoptionally substituted with at least one substituent R¹⁶; R¹⁶ ishalogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheterocyclyl, oxo, —CN, —OR^(a), —NR^(a)R^(b), —COR^(a), —CO₂R^(a),—CONR^(a)R^(b), —C(═NR^(a))NR^(b)R^(c), —NR^(a)COR^(b),—NR^(a)CONR^(a)R^(b), —NR^(a)CO₂R^(b), —SO₂R^(a), —SO₂aryl,—NR^(a)SO₂NR^(b)R^(c), or —NR^(a)SO₂R^(b), wherein R^(a), R^(b), andR^(c) are independently hydrogen, halogen, substituted or unsubstitutedalkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted alkynyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocyclyl, wherein (R^(a)and R^(b)), and/or (R^(b) and R^(c)) together with the atoms to whichthey are attached, can form a ring selected from cycloalkyl, saturatedor unsaturated heterocycle, aryl, and heteroaryl rings.
 32. Thepharmaceutical combination of claim 31, wherein the targeted therapyagent is an anti-CD20 antibody.
 33. The pharmaceutical combination ofclaim 32, wherein the targeted therapy agent is an monoclonal antibody.34. The pharmaceutical combination of claim 32, wherein the targetedtherapy agent is is selected from rituximab, ibritumomab tiuxetan,tositumonmab, ofatumumab or obinutuzumab.
 35. A pharmaceuticalcombination for use in the prevention, delay of progression or treatmentof cancer, comprising a Btk inhibitor, and an immune checkpointinhibitor, wherein the Btk inhibitor is a compound of Formula (I),

a stereoisomer thereof, or a pharmaceutically acceptable salt thereof,wherein: A is a 5- or 6-membered aromatic ring comprising 0-3heteroatoms of N, S or O; each W is independently —(CH₂)— or —C(O)—; Lis a bond, CH₂, NR¹², O, or S; S/D is a single or double bond, and whena double bond, R⁵ and R⁷ are absent; m is 0, or an integer of 1-4; n is0, or an integer of 1-4, wherein when n is more than 1, each R² may bedifferent; p is 0, or an integer of 1-2, wherein when p is 0, m isnon-zero, and when p is more than 1, each R⁶ and each R⁷ may bedifferent; R¹, R⁴, R⁵, R⁶, and a R⁷ are each independently H, halogen,heteroalkyl, alkyl, alkenyl, cycloalkyl, aryl, saturated or unsaturatedheterocyclyl, heteroaryl, alkynyl, —CN, —NR¹³R¹⁴, —OR¹³, COR¹³, —CO₂R¹³,—CONR¹³R¹⁴, —C(═NR¹³)NR¹⁴R¹⁵, —NR¹³COR¹⁴, —NR¹³CONR¹⁴R¹⁵, —NR¹³CO₂R¹⁴,—SO₂R¹³, —NR¹³SO₂NR¹⁴R¹⁵, or —NR¹³SO₂R¹⁴, wherein the alkyl, alkenyl,alkynyl, cycloalkyl, heteroaryl, aryl, and saturated or unsaturatedheterocyclyl are optionally substituted with at least one substituentR¹⁶, wherein (R⁴ and R⁵), or (R⁴ and R⁶), or (R⁶ and R⁷), or (R⁶ and R⁶when p is 2), together with the atoms to which they are attached, canform a ring selected from cycloalkyl, saturated or unsaturatedheterocycle, aryl, and heteroaryl rings optionally substituted with atleast one substituent R¹⁶; R² is halogen, alkyl, —S-alkyl, —CN,—NR¹³R¹⁴, —OR¹³, —COR¹³, —CO₂R¹³, —CONR¹³R¹⁴, —C(═NR¹³)NR¹⁴R¹⁵,—NR¹³COR¹⁴, —NR¹³COR¹⁴, —NR¹³CONR¹⁴R¹⁵, —NR¹³CO₂R¹⁴, —SO₂R¹³,—NR¹³SO₂NR¹⁴R¹⁵, or —NR¹³SO₂R¹⁴; R¹² is H or lower alkyl; R¹³, R¹⁴ andR¹⁵ are each independently H, heteroalkyl, alkyl, alkenyl, alkynyl,cycloalkyl, saturated or unsaturated heterocyclyl, aryl, or heteroaryl;wherein (R¹³ and R¹⁴), and/or (R¹⁴ and R¹⁵) together with the atom(s) towhich they are attached, each can form a ring selected from cycloalkyl,saturated or unsaturated heterocycle, aryl, and heteroaryl ringsoptionally substituted with at least one substituent R¹⁶; R¹⁶ ishalogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheterocyclyl, oxo, —CN, —OR^(a), —NR^(a)R^(b), —COR^(a), —CO₂R^(a),—CONR^(a)R^(b), —C(═NR^(a))NR^(b)R^(c), —NR^(a)COR^(b),—NR^(a)CONR^(a)R^(b), —NR^(a)CO₂R^(b), —SO₂R^(a), —SO₂aryl,—NR^(a)SO₂NR^(b)R^(c), or —NR^(a)SO₂R^(b), wherein R^(a), R^(b), andR^(c) are independently hydrogen, halogen, substituted or unsubstitutedalkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted alkynyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocyclyl, wherein (R^(a)and R^(b)), and/or (R^(b) and R^(c)) together with the atoms to whichthey are attached, can form a ring selected from cycloalkyl, saturatedor unsaturated heterocycle, aryl, and heteroaryl rings.
 36. Thepharmaceutical combination of claim 35, wherein the immune checkpointinhibitor is an inhibitor of PD-1, PD-L1, PD-L2 TIM-3, Gal-9, CTLA-4,CD80, CD86, A2AR, B7-H3, B7-H4, BTLA, BTLA, HVEM, IDO1, IDO2, TDO, LAG3,VISTA, KIR, 2B4, CD2, CD27, CD28, CD30, CD40, CD90, CD137, CD226, CD276,DR³, GITR, ICOS, LAIR1, LIGHT, MARCO, PS, OX-40, SLAM TIGHT, CTCNI, or acombination thereof.
 37. The pharmaceutical combination of claim 35,wherein the immune checkpoint inhibitor is an inhibitor of PD-1.
 38. Thepharmaceutical combination of claim 37, wherein the immune checkpointinhibitor is a monoclonal antibody.
 39. The pharmaceutical combinationof claim 37, wherein the immune checkpoint inhibitor is an antibodyselected from nivolumab, or pidilizumab.
 40. The pharmaceuticalcombination of claim 37, wherein the immune checkpoint inhibitor is amonoclonal antibody or a fragment thereof, comprising a heavy chainvariable region (Vh) amino acid sequence of SEQ ID No 24, a light chainvariable region (V1) amino acid sequence of SEQ ID No 26, and a IgG4constant domain amino acid sequence of SEQ ID NO
 88. 41. Thepharmaceutical combination of claim 31 or 35, wherein the cancer ishematologic cancer.
 42. The pharmaceutical combination of claim 41,wherein the hematologic cancer is selected from a leukemia, a lymphoma,a myeloma, a non-Hodgkin's lymphoma (NHL), a Hodgkin's lymphoma (HL), ora B-cell malignancy.
 43. The pharmaceutical combination of claim 42,wherein the B-cell malignancy is chronic lymphocytic leukemia (CLL),small lymphocytic lymphoma (SLL), follicular lymphoma (FL), mantle celllymphoma (MCL), marginal zone lymphoma (MZL), Waldenstrommacroglobulinemia (WM), Hairy cell leukemia (HCL), Burkitt's-likeleukemia (BL), B cell prolymphocytic leukemia (B-PLL), diffuse large Bcell lymphoma (DLBCL), germinal center B-cell diffuse large B-celllymphoma (GCB-DLBCL), non-germinal center B-cell diffuse large B-celllymphoma (non-GCB DLBCL), DLBCL with undetermined subtype, primarycentral nervous system lymphoma (PCNSL), secondary central nervoussystem lymphoma (SCNSL) of breast or testicular origin, or a combinationof two or more thereof.
 44. The pharmaceutical combination of claim 43,wherein the DLBCL is activated B-cell diffuse large B-cell lymphoma(ABC-DLBCL), GCB-DLBCL or Non-GCB DLBCL.
 45. The pharmaceuticalcombination of claim 44, wherein the B-cell malignancy is chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), B cellprolymphocytic leukemia (B-PLL), non-CLL/SLL lymphoma, follicularlymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL),Waldenstrom's macroglobulinemia (WM), multiple myeloma, or a combinationthereof.
 46. The pharmaceutical combination of any one of claims 43-44,wherein the B-cell malignancy is a relapsed or refractory B-cellmalignancy.
 47. The pharmaceutical combination of claim 46, wherein therelapsed or refractory B-cell malignancy is diffuse large B-celllymphoma (DLBCL).
 48. The pharmaceutical combination of claim 47,wherein the relapsed or refractory DLBCL is activated B-cell diffuselarge B-cell lymphoma (ABC-DLBCL), GCB-DLBCL or Non-GCB DLBCL.
 49. Thepharmaceutical combination of claim 45, wherein the relapsed orrefractory B-cell malignancy is chronic lymphocytic leukemia (CLL),small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia(B-PLL), non-CLL/SLL lymphoma, follicular lymphoma (FL), mantle celllymphoma (MCL), marginal zone lymphoma (MZL), Waldenstrom'smacroglobulinemia (WM), multiple myeloma, or a combination thereof, or acombination thereof.
 50. The pharmaceutical combination of any one ofclaims 43-45, wherein the B-cell malignancy is a metastasized B-cellmalignancy.
 51. The pharmaceutical combination of claim 50, wherein themetastasized B-cell malignancy is diffuse large B-cell lymphoma (DLBCL),chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Bcell prolymphocytic leukemia (B-PLL), non-CLL/SLL lymphoma, follicularlymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL),Waldenstrom's macroglobulinemia (WM), multiple myeloma, or a combinationthereof.
 52. The pharmaceutical combination of claim 31 or 35, whereinthe cancer is a sarcoma, or carcinoma.
 53. The pharmaceuticalcombination of any one of claim 31, 35 or 52, wherein the cancer isselected from bladder cancer, breast cancer, colon cancer,gastroenterological cancer, kidney cancer, lung cancer, ovarian cancer,pancreatic cancer, prostate cancer, proximal or distal bile duct cancer,and melanoma.
 54. The pharmaceutical combination of claim 52, whereinthe cancer is a relapsed or refractory (R/R) cancer.
 55. Thepharmaceutical combination of claim 54, wherein the relapsed orrefractory cancer is selected from bladder cancer, breast cancer, coloncancer, gastroenterological cancer, kidney cancer, lung cancer, ovariancancer, pancreatic cancer, prostate cancer, proximal or distal bile ductcancer or melanoma.
 56. The pharmaceutical combination of claim 52,wherein the cancer is a metastasized cancer.
 57. The pharmaceuticalcombination of claim 56, wherein the metastasized cancer is selectedfrom bladder cancer, breast cancer, colon cancer, gastroenterologicalcancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer,prostate cancer, proximal or distal bile duct cancer, or melanoma. 58.The pharmaceutical combination of claim 31 or 35, wherein the BTKinhibitor is(S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetra-hydropyrazolo[1,5-a]pyrimidine-3-carboxamide,or a pharmaceutically acceptable salt thereof.